Anal. Chem. 2007, 79, 9539-9546
Separation of Plant Pathogens from Different Hosts and Tissues by Capillary Electromigration Techniques Marie Horka´,*,† Jaroslav Horky´,‡ Hana Matousˇkova´,‡ and Karel S ˇ lais†
Institute of Analytical Chemistry, v. v. i., Academy of Sciences of the Czech Republic, Veverˇ´ı 97, 602 00 Brno, Czech Republic, and Division of Diagnostics, State Phytosanitary Administration, Sˇ lechtitelu˚ 23, 77900 Olomouc, Czech Republic
In this contribution capillary isoelectric focusing and capillary zone electrophoresis were applied for the separation and detection of different plant pathogens including Pseudomonas syringae pv. syringae, P. syringae pv. lachrymans, Pseudomonas savastanoi pv. fraxinus, P. savastanoi pv. olea, Agrobacterium tumefaciens, A vitis, Xanthomonas arboricola pv. juglandis, X. campestris pv. zinniae, and Curtobacterium sp.. The UV detection and sensitive fluorescence detection of the native phytopathogens or those dynamically modified by the nonionogenic fluorescent tenside based on pyrenebutanoate were used. The isoelectric points of the labeled phytopathogens were found comparable with the pI of the native compounds. No influence of the hosts on pIs of the strains of the genus Pseudomonas was observed. The identification of plant pathogens by gas chromatographic analysis of fatty acid methyl esters was compared with results of capillary isoelectric focusing. Capillary electromigration was successfully applied for the separation of microbes directly from plant tissue suspensions. The understanding of the plant pathology is the first line of the defense against plant diseases. Plant microorganisms, MOs, often belong to the disease-causing organisms. The identification of unknown plant pathogens remains a critical step. The methods used for these purposes should be rapid, sensitive, and accurate; they must characterize multiple representative strains of a given type of species of the genus according to their properties. Therefore, multiple complementary tests for a reliable identification are necessary to develop.1 Until now, a number of commercial systems for identification of phytopathogenic pathovars of bacteria use a variety of methods1 including fatty acid methyl esters analysis by gas chromatography, immunodiagnostic methods (ELISA, immunofluorescence, immunomagnetic separations), flow cytometry, phenotypic tests, or genotypic characterization2 such as polymerase chain reaction * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: (++420)-5-41212113. † Academy of Sciences of the Czech Republic. ‡ State Phytosanitary Administration. (1) Alvarez, A. M. Annu. Rev. Phytopathol. 2004, 42, 339-366. (2) Buyer, J. S. J. Microbiol. Methods 2002, 48, 259-265. 10.1021/ac701718v CCC: $37.00 Published on Web 11/13/2007
© 2007 American Chemical Society
(PCR).3 These chemical methods are based almost on cultivation of isolates on special agar media, which needs extra time, at least 24 h. The identification of bacteria by gas chromatographic analysis of fatty acid methyl esters4 as well as biochemical tests on microtitration plates5 (System BIOLOG, Biolog, Inc., CA) need time-consuming isolation from plant tissues. Until now these methods have not yet entirely replaced traditional culture and phenotypic tests in microbiology. New identifying and characterizing methods of plant MOs will be necessary to search in the future. The capillary electromigration techniques appear to be promising and efficient techniques for the separation of amphoteric bioparticles including microorganisms.6-10 Most often, capillary electrophoresis, CZE, and capillary isoelectric focusing, CIEF, are used. In CZE separation the properties of MOs can be characterized according to their electrophoretic mobilities, which are dependent on the background electrolytes, BGE, composition. In CIEF separation MOs can be characterized by their isoelectric points, which seem to be a more appropriate parameter for the predicting of the surface properties of the cells11 than the electrophoretic mobilities. Of course, especially at the trace analysis of MOs, some difficulties such as the adsorption of MOs onto the capillary wall,12-14 the sensitivity of the detection, and the interactions between MOs and the additives in buffer solution12,15-18 are necessary to solve. The adsorption of cells can (3) Schaad, N. W.; Frederick, R. D.; Shaw, J.; Schneider, W. L.; Hickson, R.; Petrillo, M. D.; Luster, D. G. Annu. Rev. Phytopathol. 2003, 41, 305-324. (4) Dawyndt, P.; Vancanneyt, M.; Snauwaert, C.; De Baets, B.; De Meyer, H.; Swings, J. J. Microbiol. Methods 2006, 66, 410-433. (5) O’Connell, S.; Lawson, R. D.; Watwood, M. E.; Lehman, R. M. J. Microbiol. Methods 2000, 40, 213-220. (6) Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J. Anal. Chem. 1999, 71, 5465-5469. (7) Kenndler, E.; Blaas, D. TrAC, Trends Anal. Chem. 2001, 20, 543-551. (8) Shen, Y.; Berger, S. J.; Smith, R. D. Anal. Chem. 2000, 72, 4603-4607. (9) Horka´, M.; Ru˚zˇicˇka, F.; Horky´, J.; Hola´, V.; Sˇlais, K. J. Chromatogr., B 2006, 841, 152-159. (10) Horka´, M.; Ru˚zˇicˇka, F.; Hola´, V.; Sˇlais, K., Anal. Bioanal. Chem. 2006, 385, 840-846. (11) Rijnaarts, H. H. M.; Norde, W.; Lyklema, J.; Zehnder, A. J. B. Colloids Surf., B 1995, 4, 191-197. (12) Zhao, Z.; Malik, A.; Lee, M. L. Anal. Chem. 1993, 65, 2747-2752. (13) Huang, M.; Bigelow, M.; Byers, M. LC-GC Int. 1996, 9, 658-664. (14) Ren, X.; Liu, P. Z.; Lee, M. L. J. Microcolumn Sep. 1996, 8, 529-534. (15) Corradini, D. J. Chromatogr., B 1997, 699, 221-256. (16) Rabilloud, T. Electrophoresis 1996, 17, 813-829. (17) Szo ¨ko ¨, E. Electrophoresis 1997, 18, 74-81. (18) Yao, X.-W.; Regnier, F. E. J. Chromatogr. 1993, 632, 185-193.
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be prevented and the peak shape can be improved by use of permanent capillary coating19 or by use of the additives such as poly(ethylene glycol) (PEG)6,9,20-23 which have been used also as the nonbonded coating for the suppression of electroosmotic flow (EOF).24 The sensitivity of the detection can be increased by the application of fluorometric detection. However, that is dependent mainly on tagging the bioanalytes by fluorophores.25,26 From the viewpoint of CIEF the respective isoelectric points, pIs, observed should not change significantly27,28 by labeling. The usefulness of these electromigration techniques in the field of the microbiology practice can depend on the versatility, speed of the separation, and the sensitivity of the detection. The electrokinetic characteristics of the individual microbial strains can be host-specific29 to a particular plant species, genus, or family; they can depend also on the growth phase30-32 and/or on the culture conditions.30,31 Therefore, it is necessary to gradually elucidate all these effects. This study suggests capillary electrophoretic separation of the cultured native strains of Pseudomonas (syringae pv. syringae, syringae pv. lachrymans, savastanoi pv. Fraxinus, and savastanoi pv. olea) and other plant MOs, Agrobacterium tumefaciens, including A. vitis, Xanthomonas arboricola pv. juglandis, X. campestris pv. Zinniae, and Curtobacterium sp. CIEF with UV detection or CIEF33 and/or CZE with fluorometric detection at the separation of MOs dynamically modified by the nonionogenic tenside based on pyrenebutanoate,34 namely, poly(ethylene glycol) 4-(1-pyrene)butanoate (PB-PEG), were used here. The segmental injection9,10 used here enabled reproducible results and the formation of the smooth pH gradient in the pH range of 1.8-5.533,34 at CIEF. The pH gradient is traced by suitable pI markers. The calculated isoelectric points of MOs dynamically labeled with PB-PEG were compared with the pIs of the native MOs. Since the Gram-negative Pseudomonas and certain other pseudomonads belong to the often identified plant pathogens the strains of Pseudomonas are the most widely used model for the study of pathogenesis for many other bacteria. Pseudomonas includes species pathogenic also for humans and domestic animals. Therefore, the influence of the different hosts and the different growth conditions on the analysis of the strains of (19) Horka´, M.; Planeta, J.; Ru˚zˇicˇka, F.; Sˇlais, K. Electrophoresis 2003, 24, 13831390. (20) Roosjen, A.; Karper, H. J.; van der Mei, H. C; Norde, W.; Busscher, J. Microbiology 2003, 149, 3239-3246. (21) Razatos, A.; Org, Y. L.; Boulay, F.; Elbert, D. L.; Hubell, J. A.; Sharma, M. M.; Georgiou, G. Langmuir 2000, 16, 9155-9158. (22) Kaper, H. J.; Busscher, H. J.; Norde, W. J. Biomater. Sci., Polym. Ed. 2003, 14, 313-324. (23) Desai, M. J.; Armstrong, D. W. Microbiol. Mol. Biol. Rev. 2003, 67, 38-51. (24) Preisler, J.; Yeung, E. S. Anal. Chem. 1996, 68, 2885-2889. (25) Xu, R. J.; Vidal-Madjar, C.; Se´bille, B. J. Chromatogr., B 1998, 706, 3-11. (26) Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 4887-4894. (27) Li, Y.; Buch, J. S.; Rosenberger, F.; De Voe, D. L.; Lee, C. S. Anal. Chem. 2004, 76, 742-748. (28) Sze, N. S. K.; Huang, T. M.; Pawliszyn, J. J. Sep. Sci. 2002, 25, 1119-1122. (29) Takashima, S.; Morisaki, H. Colloids Surf., B 1997, 9, 205-212. (30) Hong, Y.; Brown, D. G. Colloids Surf., B 2006, 50, 112-119. (31) Eboigbodin, K. E.; Newton, J. R. A.; Routh, A. F.; Biggs, C. A. Appl. Microbiol. Biotechnol. 2006, 73, 669-675. (32) Hayashi, H.; Seiki, H.; Tsuneda, S.; Hirata, A.; Sasaki, H. J. Colloid Interface Sci. 2003, 264, 565-568. (33) Horka´, M.; Ru˚zˇicˇka, F.; Hola´, V.; Sˇ lais, K. Electrophoresis 2007, 28, 23002307. (34) Horka´, M.; Ru˚zˇicˇka, F.; Horky´, J.; Hola´, V.; Sˇ lais, K. Anal. Chem. 2006, 78, 8438-8444.
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Pseudomonas is studied. The possibility of the CIEF application at the coidentification of MOs with known isoelectric points together with gas chromatographic analysis of their fatty acid methyl esters4 is outlined. The CIEF separations of MOs directly from the suspension of the plant tissues are examined. EXPERIMENTAL SECTION CZE and CIEF, Equipment and Procedure. The capillary zone electrophoretic and capillary isoelectric focusing experiments were carried out using the laboratory-made apparatus10 at constant voltage (-) 20 kV on the side of the detector supplied by a Spellman CZE 1000 R high-voltage unit (Plainview, NY). The lengths of the fused-silica capillaries (FS), 0.05 mm i.d. and 0.25 mm o.d. (Pliva-Lachema a. s., Brno, Czech Republic) were 350 mm, 200 mm to the detector, effective volumes of the columns ∼0.4 µL, respectively. The ends of the fused-silica capillary were dipped in 3 mL glass vials with the electrodes and BGE in CZE or with the anolyte or the catholyte solutions (CaAn) in CIEF. During the CIEF experiments, the current decreased from 40 to 60 µA at the beginning of the experiment down to 3 or 6 µA at the time of detection, depending on the sampling time interval and the sample solution. The on-column UV-vis detector LCD 2082 (Ecom, Prague, Czech Republic), connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ) at the wavelength of 280 nm was used. For the fluorometric detection the PU4027 programmable fluorescence detector (Philips Scientific, Cambridge, Great Britain) was modified. The excitation wavelength, λEX, was 335 nm, the emission wavelengths,33,34 λEM, were 463 and 480 nm. The width of the detection window was 1 mm. The light absorption (optical density) of the microbial suspensions was measured using a DU series 520 UV-vis spectrophotometer (Beckmann Instruments, Palo Alto, CA) at 550 nm. The sample injection was accomplished by siphoning action obtained by elevating of the inlet reservoir on the side of the anode relative to the outlet reservoir on the side of the cathode. The height difference of the reservoirs for the sample injection, ∆h, was 100 mm, the time of injection, tinj, was from 10 to 35 s at CZE or CIEF experiments. At CIEF the segmental injection of the sample pulse was used. The clusters of the microbial cells and the sample of the plant tissue suspension contaminated with dispersed MO were disrupted by the sonification of the microbial suspension in a Sonorex ultrasound bath (Bandelin electronic, Berlin, Germany). The sonification was processed for 1 min at the temperature 30 °C and at a frequency of 35 kHz. Between the separation runs the sample suspensions were vortexed (IKA works, Inc., Wilmington, NC). The detector signals were acquired and processed with the chromatography data station Clarity (DataApex s.r.o., Praha, Czech Republic). Gas chromatography of fatty acid methyl esters analysis was accomplished according the published procedure, e.g., ref 35. The cultures were grown on trypticase soy agar (Oxoid, Hampshire, U.K.) for 24 h at 28 °C. The presence of characteristic fatty acids was compared by the SHERLOCK microbial identification system (MIDI Inc., Newark, DE) with a database of bacteria. Plant Pathogens. The strains included in this study, see Table 1, were obtained from the Czech Collection of Microorganisms (35) Buyer, J. S. J. Microbiol. Methods 2006, 67, 187-190.
Table 1. Strains of the Plant Pathogens Included in This Study abbreviation P. s. s. 1 P. s. s. 2 P. s. s. 3 P. s. s. 4 P. s. s. 5 P. s. s. 6 P. s. s. 7 P. s. s. 8 P. s. l. P. s. f. P. s. o. A. t. A. v. X. a. j. X. c. z. C.
strain Pseudomonas syringae pv. syringae
Pseudomonas syringae pv. lachrymans Pseudomonas savastanoi pv. fraxinus Pseudomonas savastanoi pv. olea Agrobacterium tumefaciens Agrobacterium vitis Xanthomonas arboricola pv. juglandis Xanthomonas campestris pv. Zinniae Curtobacterium sp.
(CCM; Brno, Czech Republic), from the Collection of Microorganisms of the Research Institute of Plant Growing (VURV, Prahas Ruzyneˇ, Czech Republic), Plantenziektenkundige Dienst (PD, Wageningen, The Netherlands), and Collection Francaise de Bacte´ries Phytopathoge´nes (CFBP, Angers, France); the isolates were from the bacteriological laboratory of division of diagnostics State Phytosanitary Administration Olomouc. Chemicals. The buffer component tris(hydroxymethyl)aminomethane, Tris, was obtained from Sigma (St. Louis, MO), ampholyte high resolution, pH 2-4, and ampholyte pH 3-4.5, 2-morpholinoethanesulfonic acid monohydrate (MES), 3-morpholino-propanesulfonic acid (MOPS), and N-[tris-(hydroxymethyl)-methyl]-3-amino-2-hydroxy-propansulfonic acid (TAPSO) were from Fluka Chemie GmbH (Buchs, Switzerland). Poly(ethylene glycol) (Mr 400, 4000, and 10 000), taurine, and 4-(1pyrene)butyric acid were from Aldrich (Milwaukee, WI). The solution of synthetic carrier ampholytes, biolyte, pH 3-10, was obtained from Bio-Rad laboratories (Hercules, CA), L- aspartic acid (Asp) was from LOBA Chemie, Wien, Austria, N-(2-acetamido)2-aminoethansulfonic acid (ACES) and 2-[4-(2-hydroxyethyl)-1piperazinyl]-ethanesulfonic acid (HEPES) were from Merck, Darmstadt, Germany. The specifications36,37 of the used spacers and simple ampholytes are described in ref 10. All chemicals were analytical grade. Poly(ethylene glycol) pyrenebutanoate, fluorescein-based pI markers, pI ) 1.8, 3.0, 4.0, 4.7, 5.5, the low-molecular pI markers, pI ) 2.0, 2.7, 3.0, 3.3, 3.65, 4.0, 4.25, 4.7, and 4-morpholinyl acetic acid (MAA)38 were synthesized in the Institute of Analytical Chemistry Academy of Sciences of the Czech Republic, v. v. i., Brno. MAA was prepared by the reaction of morpholin and chloracetic acid (Sigma, St. Louis, MO) and PB-PEG by the reaction of 4-(1-pyrene) butyric acid and PEG 400.34 (36) Hirokawa, T.; Nishino, M.; Aoki, N.; Sawamoto, Y. K. T. Y.; Akiyama, J-I. J. Chromatogr., A 1983, 271, D1-D106. (37) Acevedo, F. J. Chromatogr., A 1991, 545, 391-396. (38) Sˇ t’astna´, M.; Tra´vnı´cˇek, M.; Sˇ lais, K. Electrophoresis 2005, 26, 53-59.
source
host
isolate 47/06-GC12
roots of apricots
isolate 60/06-GC19 isolate 56/06-GC3 isolate 48/06-GC13 isolate 59/06-GC16 isolate 81/06-2-1 isolate 81/06-2.1 isolate 83/06-PL6 VURV 6016
Crataegus vulgaris apple tree Vigna sp. pear tree Zinia elegans Pelargonium zonale twig of apricot
isolate 47/06-GC20
roots of apricot
isolate 47/06-GC24
roots of apricot
CCM 2928 CFPB 3 CCM 1448 isolate 81/06-2-2
Zinia elegans
isolate 82/06-2-3
pear tree
Preparation of the Microbial Sample. Before each experiment, the strains were thawed quickly at 28 °C and cultivated on nutrient agar (Difco Voigt Global Distribution LLC, MI) at 28 °C, both for 24 or 48 h. The microbial cultures were resuspended in physiological saline solution (PSS). The concentration of the resuspended microorganisms was estimated by the measurement of the optical density of the suspension. It was measured by spectrophotometer at 550 nm, according to the calibration curve, which was defined by reference samples. These samples were prepared by resuspension of the microbial culture in PSS. The numbers of microorganisms in reference samples were measured by dilution and by plating 100 µL of suspension on Muller-Hinton’s agar (Bio-Rad). After cultivation at 28 °C for 24 h the colonies were counted. Preparation of the Plant Tissue Suspensions. Plant tissue suspension was prepared by homogenizing 1 g of leaf washed 10 min under potable water in 10 mL of sterile demineralized water with a mortar and pestle. Safety. The potentially pathogenic microorganisms were separated here at the CZE, CIEF runs. Therefore, the strong safety procedures are necessary to adhere. All operations are performed according to the instruction for the labor with infection materials. Care must be taken to avoid contact with either of these pathogens. The use of gumgloves, the disinfection of the inner and the outer surface of the capillary, etc., after its contamination by the microorganisms is platitude. Electrolyte Systems, UV Detection. At CIEF 4 × 10-2 mol L-1 sodium hydroxide and 0.1 mol L-1 o-phosphoric acid were used as the catholyte and anolyte, respectively, with addition of 1% (v/v) ethanol (EtOH) and 0.1% (w/v) PEG 4000. Electrolyte Systems, Fluorometric Detection. At CZE the background electrolyte was composed of 1.5 × 10-3 mol L-1 taurine-Tris buffer (pH 8.4), 7 × 10-5 mol L-1 PB-PEG, 3% (v/ v) EtOH, and 0.1% (w/v) PEG 4000. For CIEF experiments presented here, 2 × 10-2 mol L-1 sodium hydroxide and 0.1 mol L-1 o-phosphoric acid were used as the catholyte and anolyte, Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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respectively, with addition of 7 × 10-5 mol L-1 PB-PEG, 1% (v/ v) EtOH, and 0.75% (w/v) PEG 10 000. Before each injection the capillaries were rinsed with acetone/ ethanol mixture (10:1 v/v) for 10 min and then back-flushed with BGE (CZE) or catholyte (CIEF) for 2 min. The rinsing procedures were carried out hydrodynamically. Sample Preparation, CIEF with UV Detection. The segmental injection of the sample into the capillary9 was employed here. The sample was injected in three partsssegment of the spacers, solution of the selected simple ampholytic electrolytes dissolved in the catholyte,10 segment of the sample mixture of MOs, and the segment of the mixture of commercial carrier ampholytes, and low-molecular pI markers for the tracing of the used pH gradient in the pH range of 2-4.7. The height differences of the reservoirs at the injection of the segments were 100 mm, and tinj. of the segment of spacers was 25 s, sample segment, 10 s, and segment of carrier ampholytes and pI markers, 35 s. The second segment was composed of the suspension of plant pathogens (see Plant Pathogens paragraph), 8 × 108 cell mL-1, dissolved in water solution of 3% (v/v) EtOH, 2% (w/v) PEG 4000, and 14.6 × 10-3 mol L-1 NaCl. The injected volume of the analytes was approximately 5 nL, which represents 5 × 103 cells injected into the capillary. The third segment contained of the water solution of pI markers, 25 µg mL-1, and 5% (w/v) of synthetic carrier ampholytes, biolyte, pH 3-10, ampholyte pH 3-4.5 and pH 2-4, in the ratio 1:2:5. Sample Preparation, CIEF with Fluorimetric Detection. Similarly as in the paragraph above, the segmental injection of the sample into the capillary9 was used here. The third segment, except the mixture of commercial carrier ampholytes, was composed of pI markers, pI ) 1.8, 3.0, 4.0, 4.7, and 5.5, for the tracing of the used pH gradient in the pH range of 2-5.5. The second segment was composed of the suspension of plant pathogens at the concentration 8 × 104 cell mL-1 dissolved in water solution of 4 × 10-4 mol L-1 PB-PEG, 3% (v/v) EtOH, 0.5% (w/v) PEG 10 000, and 14.6 × 10-3 mol L-1 NaCl. Both sample mixtures were stored for 15 min at 20 °C before use. The injected volumes of the pathogens were maximum tens of cells injected into the capillary. The concentration of each pI marker in the third segment was 5 µg mL-1 in the water solution (injected amounts 0.1 ng). Sample Preparation, CZE. The cells, 8 × 104 in 1 mL, were resuspended in 2 × 10-2 mol L-1 PB-PEG, 10% (v/v) EtOH, and 14.6 × 10-3 mol L-1 NaCl. The incubation time was 15 min at 20 °C before use. The height difference of the reservoirs at the injection of the sample was 100 mm and tinj. 10 s. Therefore, the number of injected cells is identical as at CIEF with fluorometric detection. RESULTS AND DISCUSSION CIEF. The previously published results6,7,9-11,19,23,33,34,39,40 indicate that the isoelectric points of MOs could belong to their identification attributes in the future. However, only a small number of pIs of MOs have been tabulated until now. Therefore, under constant CIEF conditions, see the Experimental Section, (39) Jucker, B. A.; Harms, H.; Zehnder, A. J. B. J. Bacteriol. 1996, 178, 54725479. (40) Harden, V. P.; Harris, J. O. J. Bacteriol. 1953, 65, 198-202.
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Figure 1. Separation of the plant pathogens by CIEF with UV detection in the pH gradient of 2-4.7. Conditions: FS capillary 0.05 mm i.d., 0.25 mm o.d., length 350 mm, 200 mm to the detection cell; applied voltage (-) 20 kV; anolyte, 0.1 × 10-1 mol L-1 H3PO4, catholyte, 4 × 10-2 mol L-1 NaOH, in both electrolytes, 0.1% (w/v) PEG 4000 and 1% (v/v) EtOH; wavelength, λ ) 280 nm; segmental injection, ∆h, 100 mm; tinj, spacer segment (ref 10) (dissolved in the catholyte), 25 s, segment of the sample, 10 s, carrier ampholytes and pI markers together, 35 s; composition, segment of carrier ampholytes, 5% (w/v) solution of biolyte pH 3-10, ampholyte, pH 3-4.5 and pH 2-4 in the ratios 1:2:5. (A) pI markers, pI, 2.0, 2.7, 3.0, 3.3, 3.65, 4.0, 4.25, and 4.7; (B) see (A), sample segment of MOssP. s. l., X. a. j., X. c. z., C., P. s. o., P. s. s. 1, P. s. f., A. v., and A. t., 8 × 108 cell mL-1, resuspended in water solution of 3% (v/v) EtOH, 2% (w/v) PEG 4000, and 14.6 × 10-3 mol L-1 NaCl; pI markers, pI, 2.0, 3.65, 4.25, and 4.7; t, migration time (min); before each injection the capillaries were rinsed for 10 min with the mixture of the acetone/ethanol, 10:1 (v/v), and then back-flushed with the catholyte for 1 min.
the isoelectric points of several strains of monitored plant pathogens were necessary to determine at first. Similarly to our previous work10 we found that the shallow pH gradient (in the pH range of 2-5) is necessary for separation, resolution, and reliable determination of the isoelectric points of native plant MOs and MOs dynamically modified by PB-PEG.33,34 For the lengthening of the pH gradient the technique of the segmental injection9,10 was used, and the pI markers for the tracing of the linearity of the pH gradients were injected together with the commercial carrier ampholytes. CIEF with UV Detection, pIs of the Native Plant Pathogens. The electropherogram of the pI markers separated by CIEF with UV detection is depicted in Figure 1A. At the experimental conditions mentioned above good linearity in the range of the required pH gradient was achieved. In the second electropherogram, see Figure 1B, the sample mixture of plant pathogens, P.s. l., X. a. j., X. c. z., C., P. s. o., P. s. s. 1, P. s. f., A. v., and A. t. (each of them 8 × 108 cell mL-1) was resuspended in the solution composed of 2% (w/v) PEG 4000, 3% (v/v) EtOH, and 14.6 mol L-1 NaCl. PEG 4000 was necessary to add into the second segment
Figure 2. Influence of the cultivation times on pIs of MOs. For conditions and designations, see Figure 1. pI markers, pI, 2.0, 3.65, and 4.7; (a and e) P. s. f., (b and f) A. t., (c and g) X. a. j., (d and h) P. s. s. 3; (A) cultivation time 24 h, (B) cultivation time 48 h.
for the prevention of aggregation and adsorption of the cells onto the inner surface of the capillary. In the third segment the pI markers, pI ) 2.0, 3.65, 4.25, and 4.7, were added. Both pH gradients, see Figure 1, part A versus part B, are comparable, and hence the values of pIs of plant pathogens from Figure 1B are possible to determine reliably. Time of Cultivation. The effect of the time of the cultivation on the change of the cell surface properties, in particular the whole surface charge of the MOs as well as the values of their isoelectric points, was studied in the experiments depicted in Figure 2. The pI markers, pI ) 2.0, 3.65, and 4.7, were used for the tracing of the required pH gradient at CIEF with UV detection. The strains in Figure 2, parts A and B, (a and e) P. s. f., (b and f) A. t., (c and g) X. a. j., and (d and h) P. s. s. 3, were cultivated at 28 °C on nutrient agar (Difco) for 24 or 48 h, respectively. In both experiments, A and B, the values of pIs of monitored MOs are the same, and it seems that the time of culturing in the range from 24 to 48 h has no significant influence on the whole surface charge of these cells. Generally, the exponential growth phase ranged from 0 to 7 h under typical growth conditions and then reached a plateau, which corresponds to the stationary growth phase. Nevertheless, at the different strains the fluctuations in the electromigration properties were found41 in the range of the time of cultivation from 14 to 48 (41) Hayashi, H.; Seiki, H.; Tsuneda, S.; Hirata, A.; Sasaki, H. Colloid Interface Sci. 2003, 264, 565-568.
Figure 3. Influence of the hosts on pIs of MOs. For conditions and designations, see Figure 1. pI markers, pI, 2.0 and 4.7; P. s. s.: (a) 1, the roots of apricot, (b) 2, the hawthorn, (c) 3, the apple tree, (d) 4, the vigna, (e) 5, the pear tree, (f) 6, the Zinia elegans, (g) 7, the pelargonium (Pelargonium zonale), (h) 8, the twig of the apricot.
h. This result suggests that the incubation time substantially affects the cell surface characteristics induced by environmental and physiological conditions. Nevertheless, previous study41 suggests that the actual change in the cell surface potential is not so important. The particular research of interference of the time of cultivation on the values of pIs in the time range from 0 to 24 h would be necessary. pIs and Pseudomonas syringae pv. syringae from Different Hosts. The genus Pseudomonas belongs to the important group of bacteria in the environment. Their universal distribution suggests a considerable degree of physiological and genetic adaptability. The host’s effect on the values of pIs of the P. s. s. strains is examined in the experiments summarized in Figure 3a-h. The strains of Pseudomonas were taken from eight different hosts, see Table 1, the roots of apricot, P.s.s. 1 (Figure 3a), the hawthorn, P.s.s. 2 (Figure 3b), the apple tree, P.s.s. 3 (Figure 3c), the vigna, P.s.s. 4 (Figure 3d), the pear tree, P.s.s. 5 (Figure 3e), the Zinia elegans, P.s.s. 6 (Figure 3f), the pelargonium (Pelargonium zonale), P.s.s. 7 (Figure 3g), and the twig of the apricot, P.s.s. 8 (Figure 3h). Here, at CIEF separation with UV detection, the pH gradient was traced only by the pI markers 2.0 and 4.7 so that the results were prevented from the possible coincidences between the injected strains of P. s. s. and pI markers. According to the results from previous experiments, see Figures 1B and 2A(d) versus 2B(h), it is found that the isoelectric points of both strains, P. s. s 1 and P. s. s. 3, were determined to be the Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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Table 2. Identification of Pathogens by GC and CIEF GC sample 1 2 3
4 5
identified microorganisms Pseudomonas syringae pv. syringae Pseudomonas fluorescens Pseudomonas syringae pv. syringae Pseudomonas putida Pseudomonas syringae pv. syringae Pseudomonas savastanoi pv. fraxinus Pseudomonas syringae pv. syringae Pseudomonas viridiflava Agrobacterium tumefaciens
pI
CIEF
[%]
ref 11 or results from Figure 1
CIEF
determined microorganism
70.8
3.1 (Figure 1)
3.1
Pseudomonas syringae pv. syringae
74.7 72.8
3.6 (ref 11) 3.1 (Figure 1)
3.1
Pseudomonas syringae pv. syringae
83.3 89.8
3.2 (ref 11) 3.1 (Figure 1)
3.1
Pseudomonas syringae pv. syringae
86.2
2.9 (Figure 1)
91.6
3.1 (Figure 1)
3.1
Pseudomonas syringae pv. syringae
93.8 85.8
2.2 (Figure 1)
2.2
Agrobacterium tumefaciens
same and equal to 3.1. From comparison of the migration times of the peaks of the individual strains of P. s. s. the influence of the hosts was not here observed, see Figure 3. Therefore, we can suppose that the influence of the hosts and growth phase of MOs on the value of their isoelectric points could be negligible. Comparison of the Identification of Cultured Plant Pathogens by CIEF and by Gas Chromatography of Fatty Acid Methyl Esters. The microbial identification system for fatty acid methyl esters analysis by gas chromatography is a standard method for identification of MOs, especially of bacteria. The fatty acid composition of the unknown is compared to a library of known in order to find the closest match.35 The sensitivity of this method is limited by the quality of the blanks. As peak area decreased in the samples, the percentage of peak area contributed by contaminants increased, and the quality of the library match declined. The accuracy of the identification of the strains from the library is expressed as the probability, %. Five samples of the unknown strains were cultivated and identified by the gas chromatography method and by CIEF with UV detection. The isoelectric points of some MOs are tabulated in ref 11; the others are determined by CIEF of plant pathogens with UV detection, see Figure 1. The results are summarized in the Table 2. The unknown strains were identified after comparison of their isoelectric points with the isoelectric points of the plant pathogens from the group of “probably present” MOs identified by GC. CIEF with Fluorometric Detection. Similarly to CIEF of the human pathogens33,34 sensitive fluorometric detection is also necessary to use for analysis of plant pathogens. The linearity of the pH gradient was verified by its tracing with the fluorescent pI markers, pI ) 1.8, 3.0, 4.0, 4.7, and 5.5, see Figure 4A. Here, the emission wavelength 480 nm was selected, where both pI markers and MOs dynamically modified by PB-PEG are detected, but with lower sensitivity.33 At the separation of MOs, P. s. l., X. c. z., C., P. s. o., P. s. s. 1, A. v., and A. t. (each of them 8 × 104 cell mL-1), the sample was resuspended in 4 × 10-4 mol L-1 PBPEG, 3% (v/v) EtOH, 0.5% (w/v) PEG 10 000, and 14.6 × 10-3 mol L-1 NaCl. The pH gradient was ranged by the pI markers 1.8 and 5.5, see Figure 4B. Both pH gradients, see Figure 4, part A versus part B, are comparable. Determined pIs of separated MOs dynamically modified by PB-PEG are wholly comparable with pIs of the native MOs. 9544 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
Figure 4. Separation of the plant pathogens by CIEF with fluorometric detection in the pH gradient of 1.8-5.5. For conditions and designations, see Figure 1. Catholyte, 2 × 10-2 mol L-1 NaOH, dissolved in CaAn, 7 × 10-5 mol L-1 PB-PEG, 0.75% (w/v) PEG 10 000, and 1% (v/v) EtOH; λEX ) 335 nm, λEM ) 480 nm; (A) pI markers, pI, 1.8, 3.0, 4.0, 4.7, and 5.5; (B) see (A), sample segment composition, P. s. l., X. c. z., C., P. s. o., P. s. s. 1, A. v., and A. t. (8 × 104 cell mL-1) resuspended in the water solution of 4 × 10-4 mol L-1 PB-PEG, 0.5% (w/v) PEG 10 000, 3% (v/v) EtOH, and 14.6 mol L-1 NaCl; pI markers, pI, 1.8 and 5.5.
CZE of MOs with Fluorometric Detection. The difference between pIs of the selected plant MOs are relatively small. CZE could be the electromigration technique for the identification of MOs by the common procedures. At CZE with fluorometric detection, see Figure 5, the taurine-Tris buffer (1.5 × 10-3 mol L-1, pH 8.4) with dissolved 7 × 10-5 mol L-1 PB-PEG, 3% (v/v) EtOH, and 0.1% (v/v) PEG 4000 was used. The microbial sample was composed of 8 × 104 cell mL-1 each of the strains A. t., X. a. j., P. s. o., P. s. s. 1, P. s. l., P. s. f., and C., resuspended in 2 × 10-4 mol L-1 PB-PEG, 10% (v/v) EtOH, and 14.6 × 10-3 mol L-1 NaCl. The zones of MOs were detected at the maximum of the emission
Figure 5. CZE of the plant pathogens with fluorometric detection. For conditions and designations, see Figures 1 and 4. BGE composition, 1.5 × 10-3 mol L-1 taurine-Tris (pH 8.4), 7 × 10-5 mol L-1 PB-PEG, 0.1% (w/v) PEG 4000, and 3% (v/v) EtOH; λEM ) 463 nm; tinj., 10 s; sample composition, A. t., X. a. j., P. s. o., P. s. s. 1, P. s. l., A. v., P. s. f., and C. resuspended in the water solution of 2 × 10-4 mol L-1 PB-PEG, 10% (v/v) EtOH, and 14.6 mol L-1 NaCl.
wavelength of PB-PEG, 463 nm. Narrow peaks of the cells are detected, when PEG 4000 is dissolved in BGE. Less than 10 of MOs dynamically modified by PB-PEG were fluorometrically detected at CIEF or CZE under the optimized conditions. CIEF of MOs from the Plant Tissue Suspension with UV or Fluorometric Detection. The possibilities of the application of CIEF in the separation of pathogens direct from the model plant tissue suspensions of apricot Armeniaca species, root, with resuspended P. s. s. 1 or A. t., Mirabilis jalapa, leaf, with P. s. s. 1, A. t., or Nicotiana tabacum, lamina, with A. t., were examined in the experiments depicted in the electopherograms in Figure 6. The numbers of cells resuspended in 1 mL of the plant tissue suspension were 8 × 108 and 3 × 105 at the CIEF with UV, see Figure 6, parts A and B, or fluorometric detection, see Figure 6, parts D and E, respectively. The CIEF separation conditions were identical to CIEF with UV detection and CIEF with fluorometric detection for the appreciation of the availability of the selected CIEF procedure including the sample preparation and numbers of MOs injected into the capillary. With respect to the higher level of the background after direct injection of the blank sample of the plant tissue suspension into the capillary the linearity of the pH gradient was verified separately, see Figure 6C, the dependence of pI on the migration time t. The pH gradient was traced by the low-molecular pI markers from 2.0 to 4.7. The isoelectric points of focused pathogens, P. s. s. 1 and A. t., see Figure 6, parts A and B, can be deduced directly from dependence in Figure 6C. The values of pIs are the same as in Figure 1B. The electropherograms of CIEF with fluorometric detection are depicted in Figure 6D-F. Different from CIEF with UV detection the levels of the backgrounds are relatively low after direct injection of the blank sample of the plant tissue suspension into the capillary, see Figure 6F, curves 1 and 2ssuspension of Armeniaca sp. without or with PB-PEG in the sample, respec-
Figure 6. Separation of MOs from the plant tissue suspensions by CIEF with UV (A-C) or fluorometric detection (D-F). For conditions and designations, see Figures 1 and 4. (A) A. t. in the suspension of Armeniaca sp., root; (B) P. s. s. 1 in the suspension of Mirabilis jalapa, leaf; (C) the dependence of pI markers, pI 2.0, 2.7, 3.0, 3.3, 3.65, 4.0, 4.25, and 4.7 on t; (D) A. t., 3 × 105 cell mL-1, in the suspension of Nicotiana tabacum, lamina; pI markers, pI 1.8, 4.0, and 4.7; (E) see (D) P. s. s. 1 in the suspension of Armeniaca sp., root, ; (F) injection of the blank sample of the plant tissue suspension into the capillary; curves 1 and 2, the suspension of Armeniaca sp., root, without or with PB-PEG in the sample, respectively; curve 3, the suspension Nicotiana tabacum, lamina, with PB-PEG in the sample.
tively, and curve 3ssuspension of Nicotiana tabacum with PBPEG in the sample. Therefore, the fluorescent pI markers 1.8, 4.0, and 4.7 were added into the third segment at the injection of the sample into the capillary. According to their migration times we can estimate the linearity of the gradient and determined pIs for A. t. and P. s. s. 1. Their values are once again the same as in Figure 1B and are not influenced by the origin of the used plant tissue suspension. The sensitivity of CIEF with fluorometric detection is sufficient for the detection of plant pathogens in real samples, and there is no problem to inject the sample of the plant tissue suspension into the capillary with its i.d. of 50 µm. CONCLUSIONS Capillary electromigration methods are found to be very useful as an alternative method for the identification of plant pathogens. The values of isoelectric points of MOs were found to be not hostAnalytical Chemistry, Vol. 79, No. 24, December 15, 2007
9545
specific; they were also independent of the time of MOs cultivation in the range from 24 to 48 h. Thus, the estimated pIs of MOs were successfully used as the supporting identification attributes for the matching of the results obtained by the standard methods GC analysis of the fatty acid methyl esters currently used for the identification of MOs. CIEF with UV or fluorometric detection was found feasible for detection of pathogens in the plant tissue suspension injected directly into the separation capillary. The sensitivity of the fluorescence detection enabled analysis of several individual cells without change of the properties of the cells. The estimated isoelectric points of the plant pathogens dynamically
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modified by PB-PEG are found to be comparable to the pIs of native ones. ACKNOWLEDGMENT This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic No. IAAX00310701 and by the Institutional research plan AVO Z40310501. Received for review August 15, 2007. Accepted September 26, 2007. AC701718V
