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Letters in Applied Microbiology 1990, 10, 241-244

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli W .C. W O N GDepartment of Primary Industries, Plant Pathology Branch, Meiers Road, lndooroopilly, Queensland 4068, Australia Received 10 January 1990 and accepted 25 January 1990

W O N GW.C. , 1990. Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli. Letters in Applied Microbiology 10, 241-244.

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X . campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG,,. The reactive antigens are thermostable, but their chemical nature has not been determined.

Halo and common bacterial blights of bean (Phaseolus vulgaris L.) caused by Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli, respectively, are seedborne (Neergaard 1979). Biochemical characteristics are not very useful in distinguishing pathovars of Ps. syringae (Palleroni 1984) and pathovars of X. campestris (Bradbury 1984). T o date, a host test is the method commonly used to differentiate these pathogens from other pathovars (Lelliott & Stead 1987). However, this method for detection and identification of pathogens is not only time consuming and cumbersome but also has limited reliability. The use of hybridoma technology may provide a rapid, sensitive and reliable method for detection and identification of the bean blight bacteria. The objective of this study was to investigate the possibility of producing pathovar-specific monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (EDTA, sodium salt) extractable lipopoly-

saccharide (LPS) antigens of both halo and common blight pathogens.

Materials and Methods BACTERIAL STRAINS

Pseudomonas syringae pv. phaseolicola (strain 03 12) and Xanthomonas campestris pv. phaseoli (fuscans strain 0264) isolated by Dr M.L. Moffett from navy beans in Queensland were used for production of MAbs. Bacterial strains used to test the specificity of MAbs shown in Tables 1 and 2 were obtained from Miss B. Wood of this laboratory. Strains of Ps. syringae pv. phaseolicola and X. campestris pv. phaseoli were isolated from various bean producing areas in Queensland. Because of the stringent quarantine restrictions in Australia, strains of Ps. syringae pv. phaseolicola and X. campestris pv. phaseoli from other major bean producing countries could not be included in this study.

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W . C . Wong Table 1. Reaction of intact whole cells of bacterial strains with monoclonal antibodies (MAbs) produced to Pseudomonas syringae pv. phasealicola by an indirect ELISA method* MAbs Bacterial strain tested?

No. strain tested

A6-1

A6-2

Pseudomonas syringae pv. phaseolicola Other Pseudomonas spp. Xanthomonas spp. Erwinia spp. Corynebacterium spp. Bacillus spp. Escherichia coli Gram negative, bean epiphytes

9 26 49 3 4 4 2 10

9 0 0 0 0 0 0 0

9 0 0 0 0 0 0 0

* A result was taken as positive when O.D. at 492 nm was greater than 0.2.

t Pseudomonas spp. included Ps. caryophylli, Ps. auenae, Ps. andropoyonis, Ps. cichorii, Ps. flectans, Ps. tolaasii, Ps. solanacearum biotypes 2, 3 and 4, Ps. syringae pv. syringae, pv. glycineu, pv. pisi, pv. maculicola, pv. mori, pv. striufaciens, pv. tabaci, pv. tayetis, pv. viridiflavauo; Erwinia spp. included E. chrysanthemi, E. carotovora subsp. carotovora, subsp. atroseptica; Corynehacterium spp. included C. michiganense subsp. michiganense, subsp. insidiosum, C. flaccumfaciens subsp. flaccumfaciens; Bacillus spp. included B. subtilis, B. megaterium, B. lichenijormis and unidentified Bacillus spp. t Xanthomonas spp. included X . cumpestris, pv. campestris, pv. oryzae, pv. cucurbitae, pv. aljaljae, pv. holcicola, pv. juglandis, pv. manyiJeraeindicae, pv. pruni, pv. translucens, pv. vesicatoria, pv. vitians, pv. zinniae, pv. pelargonii, pv. dieffenbachiae, pv. begoniae and X . fragariae.

However, type strains (X. campestris pv. phaseoli, PDDCC 5834a; X . cumpestris pv. malvaceurum, PDDCC 5739a; Ps. avenue, PDDCC 3183; P s. caryophylli, PDDCC 512a and Ps. syringae pv. phaseolicola, PDDCC 2740a) obtained from the Plant Diseases Division Culture Collection (PDDCC), DSIR, Private Bag, Auckland, New Zealand were included.

P R E P A R A T I O N OF A N T I G E N S

Bacterial cultures were grown for 48 h in nutrient broth (Oxoid CM1) at 25°C. Extraction procedures for LPS using EDTA were performed essentially as described by Leive et al. (1968). The crude LPS adjusted in sterile normal saline to 0.25 mg/ml was used as an immunogen

Table 2. Reaction of intact whole cells of bacterial strains with a monoclonal antibody (MAb) produced to Xanthomonas campestris pv. phaseoli (fuscans strain) by an ELISA method* Bacterial strain testedt

No. strain tested

MAb XP2

Xanthomonas campestris pv. phaseoli pv. phaseoli (fuscans strain) pv. maloacearum Other Xanthomonas spp. Pseudomonas spp. Erwinia spp. Corynebacterium spp. Bacillus spp. Escherichia coli Gram negative, bean epiphytes

16 2 9 22 34 3 0 2 2 10

16 2 9 0 0 0 0 0 0 0

* A result was taken as positive when O.D. at 492 nm was greater than 0.2. t See footnote to Table 1.

MAbs to Ps. syringae and X. campestris pathouars without further treatment. Whole cells washed once in distilled water were used for all screening test systems.

IMMUNIZATION A N D HYBRIDOMA PRODUCTION

Balb/c mice were immunized subcutaneous~y with 0.2 ml antigens without adjuvant on day 1 and day 21, and an intraperitoneal (i.p,) inoculation on day 40. O n day 60, 3 d before fusion, the mice were given a final i.p. booster. Spleen cells from the immunised animals Were fused with P3-63/Ag8.653 mouse myeloma cells using conditions previously described (Wong et a / . 1988). Specific MAbs detected by the Petri dish enzyme immunoassay method and subcloned twice with limiting dilutions were tested further against a wide range of bacterial isolates (Tables 1 and 2) using the ELISA and indirect immunofluorescence systems.

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taining 0.001% Tween 20. Reactive sites were blocked by adding 1% bovine serum albumin (BSA) containing 0.1 mol/l glycine in PBS. Culture supernatant fluid was used undiluted. A 1 : 400 dilution of goat anti-mouse IgG peroxidase conjugate (Cappel Laboratories, Malvern, PA, USA) was used to detect MAb. The plate was washed three times after each incubation for 30 min. Substrate (0.1% o-phenylenediamine in citrate phosphate buffer, PH 4.5, containing o.oo6'x H,od to react for 5 min at room temperature, was stopped by adding 2N H,SO, solution. The absorbance of the soluble product was measured at 492 nm. INDIRECT IMMUNOFLUORESCENCE (IF)

A 10pl aliquot of bacterial suspension (105 cfu/ml) in distilled water applied to a microscopic slide was in methanol at - 200c for min, It was washed twice in distilled water and air dried. The IF staining procedures were performed as described by Wong et al. (1988).

PETRI DISH ENZYME IMMUNOASSAY (PEIA)

ISOTYPING

The PEIA method (Wong et al. 1988) was used in this study with minor modifications. Prior to the addition of culture supernatant fluid, each spot was coated with a 1Opl aliquot of bacterial suspension containing approximately 106 cfu/ml in distilled water. Spots were air dried overnight, fixed for 15 min at room temperature in 0.25% glutaraldehyde solution, washed twice in distilled water and again allowed to air dry. All subsequent steps were performed as previously described. These modifications allowed the detection of specific MAbs by a selective panel of antigens in the initial screening tests.

INUIKI'C'T E N Z Y M E - L I N K E D IM M

u N O S O K HI: N T

ASSAY

(E L I S A )

Poly-L-lysine (PLL) solution (Sigma, 0,001 g/lOO ml phosphate buffered saline (PBS), pH 7.2) was placed into each well of a 96-well microplate (Nunc, Roskilde, Denmark) and incubated for 1 h at room temperature. The PLL solution was removed and the microplate coated with a bacterial suspension in PBS (with concentration corresponding to O.D. at A600 nm = 0.1). The plate, incubated overnight at 3 7 T , was fixed in 0.25?4 glutaraldehyde solution and then washed three times in PBS con-

The class and subclass of antibodies were determined by isotYPinf3 kit assays according to the instructions given by the manufacturer (Commonwealth Serum Laboratories, Parkville, ViC., Australia). Results and Discussion

Of the produced and tested, only three (Tables 1 and 2) were useful for serotyping ps, syringae pv. phaseolicola and campestris pv, phaseoli. MAbs (A6-1 and A6-2) produced to Ps. syringae pv. phaseolicola were pathovar specific (Table 1). MAb XP2 (Table 2) produced to X . campestris pv. phaseoli reacted with all isolates of this pathovar (including fuscans strains) as well as with X . campestris pv. malvacearum, the causal organism of cotton leaf blight, independent of strain and source; it did not cross react with any other known bacteria or unidentified Gram negative epiphytes obtained from navy bean seed or leaves. Using the ELISA method with MAb XP2, no quantitative differences in reactivity were detected between X . campestris pv. phaseoli and X . campestris pv. maluacearum. The specificity of MAbs XP2, A6-1 and A6-2 was further confirmed by the IF method. Posi-

x.

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w.c. wong

tive reactions gave a strong, bright, blue-green fluorescence over the entire periphery of the bacterial cell wall. Results presented here indicate that there were pathovar specific antigens present in the EDTA-LPS extract prepared from Ps. syringae pv. phaseolicola whereas that from X . campestris pv. phaseoli contained a cross-reacting antigen which is specifically shared between the two X . campestris pathovars. This suggested that production of a pathovar specific MAb to X . campestris pv. phaseoli may be difficult to achieve with an EDTA extract. However, such shared specificity is an indication of their evolutionary relationship as these two pathovars are closely related (Bradbury 1984; Logan 1960). Results also demonstrate that those specific and crossreactive determinants are antigenically conserved in all reactive bacterial strains although the chemical nature of these antigenic determinants has not been determined. MAbs XP2 and A6-1 were both found to possess the IgG3 heavy chain whereas the isotype of MAb A6-2 was IgG,,. Heating of whole cells of X . campestris pv. phaseolicola, X . campestris pv. maloacearum, and Ps. syringae pv. phaseoli (including fuscans strains) at 100°C for 2 h in a water bath had no adverse effect on the reactivity of the cell wall antigens with these MAbs. This indicates that the reactive antigens are thermostable. The major advantage of the MAb XP2 produced in this study is that it can detect both X . campestris pv. phaseoli and X. campestris pv. malvacearum. Furthermore, both of these path-

ovars are host specific (Logan 1960) and it is highly unlikely that the former would be found in cotton or the latter in navy bean seed. It is thus anticipated that MAb XP2 can be used for the dual purposes of detecting and identifying X . campestris pv. phaseoli in navy bean seed and X . campestris pv. maloacearum in cotton seed. Further work is in progress to study the chemical nature of these antigenic determinants as well as to evaluate the potential of these MAbs for routine seed health testing.

References BRADBURY, J.F. 1984 Xanthomonas Dowson 1939. In Bergey S Manual of Systematic Bacteriology Vol. 1, ed. Krieg, N.R. & Holt, J.G. pp. 199-210. Baltimore, MD: Williams & Wilkins. LEIVE,L. SHOVLIN, V.K. & MERGENHAGEN, S.E. 1968 Physical, chemical, immunological properties of lipopolysaccharide released from Escherichia coli by ethylenediamine tetraacetate. Journal of Biological Chemistry 243,6384-639 1. LELLIOTT, R.A. & STEAD,D.E. 1987 Methods for the diagnosis of bacterial diseases of plants. In Methods in Plant Pathology Vol. 2, ed. Preece T.F. pp. 152168. London: Blackwell Scientific Publications. LOGAN,C. 1960 Host specificity of two Xanthomonas species. Nature 188,479480. NEERCAARD, P. 1979 Seed Pathology Vol. 1. London: Macmillan Press. PALLERONI, N.J. 1984 Pseudomonas Migula 1894. In Bergey’s Manual of Systematic Bacteriology Vol. 1, ed. Krieg, N.R. & Holt, J.G. pp. 141-172. Baltimore, M D : Williams & Wilkins. WONG,W.C., WHITE,M. & WRIGHT,1.G. 1988 Production of monoclonal antibodies to Fusarium oxysporum f.sp. cuhense race 4. Letters in Applied Microbiology 6, 3 9 4 2 .