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Enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) in the serodiagnosis of plant pathogenic bacteria. G. LAZAROVITS. Agricultural ...
Enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) in the serodiagnosis of plant pathogenic bacteria G. LAZAROVITS Agricultural Canada, Research Centre, U n i v e r s i ~Sub Post OfjLice, London, Ont., Canada N6A 5B7 AND

D. ZUTRAA N D M. BAR-JOSEPH

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Department of' Plant Pathology, Agricultural Research Organization, The Volcani Centre, Bet Dagan , Isrczel Received July 7 , 1986 Accepted October 22, 1986 LAZAROVITS, G., D. ZUTRA, and M. BAR-JOSEPH. 1987. Enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) in the serodiagnosis of plant pathogenic bacteria. Can. J . Microbiol. 33: 98- 103. The usefulness of enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) for diagnosis and identification of plant pathogenic bacteria was tested.Five pathovars of Xrinthomonas campestris and two antisera, one produced against pv. vesicatoria and the other against pv. translucens, were used in a model system. A 10-min incubation of the bacterial cells, dot blotted on membranes, in diluted sera, followed by either alkaline phosphatase conjugated protein A or goat antirabbit globulin, resulted in a specific reaction between the homologous serum and bacteria. Populations of 1000-2000 cfu per spot (ca. 0.3 cm2) could be detected with these reagents. The streptavidin-biotinylated peroxidase complex produced a definitive reaction with as few as 800 cfu, but cross-reactions became evident at the higher cell concentrations among all five pathovars in tests with both antisera. Cell-free extracts, obtained by centrifugation of boiled bacteria, reacted similarly to live cells. Unrelated bacteria did not react with either antiserum. Extracts of lesions from tomato and pepper leaves infected with X. campestris pv. vesicatoriu reacted positively with the antiserum produced against this pathovar but not that produced with pv. translucens. Samples of supernatants from boiled lesions reacted with similar intensity as those from homogenized tissues.

LAZAROVITS, G . , D. ZUTRAet M. BAR-JOSEPH. 1987. Enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) in the serodiagnosis of plant pathogenic bacteria. Can. J . Microbiol. 33 : 98-103. Nous avons virifit l'utilite d'une mithode d'immunoessai enzymatique sur membranes de cellulose (ELISA en points) pour le diagnostic et I'indentification de bactiries phytopathogenes. Dans notre modele d'etude, nous avons utilisi cinq pathovariants (pv.) de Xanthomonus campestris et deux immunsirums, l'un dirigi contre le pv. vescatoria et l'autre contre le pv. translucens. Apres inoculation en points de cellules bacteriennes et incubation pendant 10 min en prisence de sirurn dilui, on traite par la protiine A conjuguie a la phosphatase alcaline ou une globuline de chkvre anti-lapin et on obtient une riaction spicifique entre le sirum homologue et les batteries. De cette f a ~ o nil est possible de ditecter des populations de 1000-2000 cfu par point ( c a . 0,3 cm2). Le complexe streptavidine - biotinyl-peroxidase donne une reaction positive definitive avec aussi peu que 800 cfu, mais des riactions croisies apparaissent a des concentrations cellulaires plus ilevies et ce avec les cinq pathovariants et les deux antiserums. Les extraits bactiriens bruts obtenus par centrifugation de bactiries porties a I'ibullition ont donni les m6mes riactions que les cellules vivantes. Des bactiries non-apparenties n'ont pas donne de reaction avec I'un ou l'autre des immunserums. Des extraits obtenus de lisions recueillies sur des feuilles de tomate et de poivirir infecties avec X. campestris pv. vesicatoria donnaient des riactions positives avec I'immunsirum produit contre ce variant pathogkne mais pas avec I'imrnunsirum produit contre pv. translucens. Des surnageants de lesions bactiriennes parties a l'ibullition donnaient des riactions dont l'intensiti itait la m2me que celle obtenue avec du tissu homogineise. [Traduit par la revue]

Introduction Enzyne-linked immunosorbent assays (ELISA) are most commonly performed using polystyrene plates as the adsorbent matrix (Clark and Bar Joseph 1984). Recently, materials such as nitrocellulose and plastic membranes were found to be acceptable, and often superior, as alternative solid-phase components (Towbin and Gordon 1984; Banttari and Goodwin 1985; Hawkes et al. 1982). Immunoassays on such membranes, termed dot immunobinding (Towbin and Gordon 1984) or dot-ELISA (Banttari and Goodwin 1985), generally involve applying microlitre quantities of sample to the adsorbing membrane and then exposing the membranes to a solution containing antibody. This is then followed by an enzymeconjugated material that reacts specifically with the antibody. Enzymatic activity is detected with an appropriate substrate which, after conversion, precipitates either directly or indirectly to form a c~louredspot. This technique, although used extensively in basic biochemical research and found to be both sensitive and versatile (Towbin and Gordon 1984), has been tested only to a limited extent for plant disease diagnosis, where it was used mainly for

detection of plant viruses (Banttari and Goodwin 1985; P. H. Berger, D. W. Thornbury, and T. P. Pirone. 1984. Phytopathology, 74 (Abstr.); D. J. Gumpf, W. Kositratana, and G. Y. Zheng. 1984. Phytopathology, 74: 847 (Abstr.); C. A. Powell. 1984. Phytopathology, 74: 847 (Abstr.)). In tests for potato viruses S , X, and Y, dot-ELISA was found more sensitive, easier to perform, and less expensive than conventional ELISA (Banttari and Goodwin 1985). Microbiological membrane filters have been in use for some time for detection of bacteria using fluorescent brighteners or immunofluorescent probes (A. Trigalet , R . Samson, and A. Coleno. 1979. Proc. Int. Conf. Plant Pathog. Bact., 4th. pp. 271-288; H. Y. Domen and A. M . Alvarez. 1979. Proc. Int. Conf. Plant Pathog. Bact., 4th. pp. 301 -305). Dot-ELISA bears very close similarity to this assay but has the advantage that it eliminates the requirement for a iluorescent microscope. The applicability of dot-ELISA for diagnosis of plant diseases caused by bacteria was recently compared with other immunodiagnostic procedures by Malin et al. (1985) who found it most useful for identification of Xanthornonus carnpestris pv. phuseoli. In the present study we also examined the potential of

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dot-ELISA for immunocharacterization of bacteria obtained from cultures and from diseased plant tissues. Selected pathovars from the Xanthomonas campestris group of bacteria were used as a model system.

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Materials and methods Cultural conditions Cultures of Xanthomonas campestris pathovars were from the collection of D. Zutra and were cultured on nutrient agar at 25OC. The isolate X. campestris pv. campestris was originally isolated from cauliflower, pv. vesicatoria from pepper, pv. translucens from wheat, pv , pelargonium from geranium, and pv . begonia from begonia. Erwiniu carotovora , Corynebac.terium fcisc*ia, and Pseudomonus syringue pv . luchrymans were pathogenic isolates from potato, pelargonium, and cucumber, respectively, and were also cultured on nutrient agar. Preparation of antisera Bacterial cells of Xanthomonas curnpestris pv. vesic.crtoria and pv. translucens were grown on nutrient agar (Difco) at 28°C overnight. The cells were washed with and suspended in phosphate-buffered saline (PBS), pH 7.4. Cells, at a concentration of about 2 x 10' cfu/mL in PBS, were injected intravenously into New Zealand white rabbits at 4-day intervals starting with a 0 . 1 mL for the first injection, followed by gradual increase in dosage of 0.5, 1 .O, 2.0, 3 .O, 4.0, and 5.0 mL. The rabbits were bled 30 days after the first injection. Serum titers were examined using the tube agglutination test at 37OC and sera with titers above 1 / 10 000 were pooled and stored with thiomersal ( l o p 4 M) (British Drug House, England) at - 20°C. Dot-ELISA The protocol described is that used routinely and one that has been found to be most successful in assays of several different bacterial genera (Zutra et a1 . 1986). Nitrocellulose membranes (NCM) (Schleicher and Schuell, Dansell, West Germany, 0.45 pm pore size) were cut to the size required for the experiment with a razor blade and marked with pencil into an 8 x 8 mm grid. The NCM were rinsed in distilled water. followed by Tris-buffered saline (TBS; 50 mM Tris (hydroxymethyl) aminomethane, 150 mM sodium chloride 1 0 - 9 thiomersal) and then they were thoroughly dried on Whatman 3 MM paper. Bacterial suspension (1- or 2-pL drops) were applied to the membranes with a Hamilton microsyringe, and after air drying, they were placed in an oven at 80°C for 30 min to stabilize binding and inactivate bacterial enzymes. Unreacted sites on the NCM were blocked by incubating the membranes for 15 min in the soluble fraction of 3% casein (w/w) (Sigma. St. Louis, MO) in TBS. This was followed by a rinse in TBS containing 0.05% Tween 20 (TBS-Tween) and further incubation for 5 min in the casein solution. The strips were then transferred to dilutions of antisera in TBS-Tween containing 1 % bovine serum albumin (w/w, Sigma) (TBS-BSA-Tween). In this study the sera were generally used at a dilution of 1 : 1000 or 1:2000 and the NCM were incubated in the antisera for 10 min. This was followed by 5 rinses with TBS-Tween at approximately 2-min intervals. Several enzyme-conjugated probes for antibody-antigen interaction were tested. Tests using alkaline phosphatase conjugated goat antirabbit globulin and alkaline phosphatase conjugated protein A (both from Bio Yeda, Rehovot, Israel) followed similar schedules. These reagents were used at 1 : 1000 dilution in TBS-BSA-Tween and incubation in these solutions was for 1.5 h. Unbound conjugates were removed by rinsing the membranes as described previously. Enzyme activity was detected by covering the strips with a solution of substrate composed of naphthol AS-MX phosphate (0.4 mg/mL) and fast red salt (6mg/mL) (both from Sigma) in 0.1 M Tris buffer, pH 8.0, containing 1 mM MgCl?. Incubation in biotin-conjugated goat antirabbit globulin (Bio Yeda) at 1:5000 (dilution in TBS-BSA-Tween) was for 30 min. After 5 rinses in TBS-Tween, the membranes were placed in a 1 :3000 dilution

+

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of streptavidin - biotinylated peroxidase complex (Amersham Int., Amersham, U.K.) in TBS-BSA-Tween for 30 min. The membranes were rinsed as previously and enzyme activity was detected in a freshly prepared mixture composed of imidazoIe (6 mg in 8 mL of H20), orthoanizide (6.0 mg in 2 mL of ethanol) (both from Aldrich Chemical Co., Milwaukee, W1) and 10 pL of 30% H202 . Incubation was continued in the substrates until a definitive reaction was observed. The strips were then rinsed in water and dried on filter paper. All incubations and washes were performed in plastic trays with gentle agitation on a platform shaker at room temperature. Diagnostic. tests On bac.teriufrom c.ultures Bacteria suspensions were prepared from 3-d-old cultures. Suspensions, in PBS, were adjusted to 5 X 10' or 10' cfu/mL from optical density measurements at 440 nm. Subsequent serial dilutions were made in PBS. Suspensions were maintained for extended periods with M thiomersal at 4OC. On supernatunts from boiled cells The effect of heat on the release of antigenic components from bacterial cells was investigated. Cell suspensions ( 1 x 10' cfu/mL) were autoclaved in sealed tubes for 30 min at 12 1°C and the cells were pelleted by centifugation at 15000 x g for 10 min. The supernatants were removed and used in subsequent tests for antigenicity . On buc~teriufrom infected tissues Pepper(cv. Maor) and tomato (cv. Faculty 16) plants (5- to 8-leaf stage) were inoculated by applying 5 x lo6 cfu/mL of X . campestris pv. vesicatoria to the abaxial surface of the leaves with a brush. A small amount of carborundum and Tween 20 was included in the suspension to facilitate bacterial infection. Leaves to be used as controls were treated with the same carrier solution without bacteria. Following inoculation, plants were incubated for 2 d under high humidity in a glass chamber in a greenhouse maintained at 25OC. After the plants were removed they were kept under the same conditions in the greenhouse for an additional 5 d. At this time bacterial lesions were obvious on both tomato and pepper leaves. One infected leaf, with fairly uniform distribution of lesions, and one healthy leaf were harvested from each of four plants of tomato and pepper. The leaves were rinsed under running tap water for approximately 5 min and were then blotted dry on paper towels. Leaf discs (5 mm diameter), with two to three bacterial lesions, were excised from each leaf and a single disc was placed into Eppendorf centrifuge tubes containing 0.2 mL H20. In one experiment, the tissues were macerated into microscopic fragments with a glass rod. In another, the tubes containing the leaf pieces were closed. placed in a boiling water bath for 20 min, and then centrifuged at 15 000 x g for 5 min. The supernatant was removed and used in assays for antigenicity. The ability to detect bacteria from single lesions was tested by placing pieces of leaf tissue, bearing one lesion, into drops of distilled water of 2 5 , 50-, 100-, and 200-pL volumes located in sterile plastic petri plates. The submerged tissues were sliced into small pieces with a razor blade and the bacteria allowed to move into the water for 1 h. The final volume of the drops were measured, and when required, adjusted to the original volume. In addition to the undiluted preparations, serial 1:5 dilutions (made with distilled water) were also assayed. Generally, 2-pL volumes were used for immunoassays. All tests were carried out at least three times and the presence of X. campestris pv. vesicatoria in the various preparations was confirmed by dilution plating on agar. Densitometn meusurements Densitometric determinations were carried out on photographs taken of the membranes (Fig. l ) , using a model 620 Video densitometer (Bio Rad, Richmond, CA). Integration of peaks was with a Hewlett Packard 3390A integrator.

Results Identification of X . campetris pathovars by dot-ELISA Strains of X. campestris pv. vesicatoria and pv. translucens were consistently differentiated by their homologous sera from

CAN. J. MICROBIOL. VOL. 33. 1987

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FIG. 1. The reaction of five Xanthomonas campestris pathovars and three unrelated bacteria genera with two antisera produced against X . campestris pv. vesiccrtoria (plates a and c.) and pv. translucens (plates b and ( I ) . Plates cr and b were incubated with alkaline phosphatase conjugated protein A (Prot. A - Alk. Phos.), and c- and d with streptavidin - biotinylated peroxidase complex (Strep. - perox.). Key to bacteria C , Psrudomorlcrs 1crc.hryrnnns;D-H, Xcrnthomoncrs c~crrnprstrispathovars: D, strains: lane A, Ersinia carotovora; B, Co~nebactrrium~ftr.sc~icrn.s; pv . vesica!oria; E, pv . pelargonium; F, pv. begonia; G , pv . trcrn.sluc~er1.s;H pv . c-c1mpr.vtri.v.

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other bacterial genera and Xunthomonus pathovars using the dot-ELISA protocol described here. Bacterial populations in excess of 2000 cfu per spot (ca. 0.3 cm2) could be readily detected when the homologous serum was followed with either alkaline phosphatase conjugated protein A (Fig. 1, plates u and b) or goat antirabbit globulin which gave identical results (not shown). Using these probes, X. campestris pv. ve.sicutoriu antiserum was found to react only with the homologous antigen (Fig. 1, plate a ) , whereas the antiserum against X. campestris pv. translucens also reacted with X. campestris pv . vesicutoriu . In both instances, however, the homologous reaction was evident whether discerned by visual inspection (Fig. I, plates u and b) or from results of densitometric measurements (Figs. 2u, and 2b). Similar reflectance values with heterologous and homologous reactions were obtained when cell numbers applied of the former exceeded the latter by a factor of 20 (X. cumpestri.~ pv. translucerzs antiserum) and 50 (X. c-umpestrispv. vesicutoria antiserum) (Figs. 2u and 2b). When the streptavidin biotinylated peroxidase complex was used for detection, as few

as 800 cfu of X. campestris pv. vesicatoria or X. campestris pv. translucens elicited a distinct homologous reaction (Fig. I , plates c and 4. The apparently greater sensitivity provided by this reagent also revealed distinct cross-reactions among all the X. campestris pathovars with both antisera tested (Fig. I , plate c and d), especially at the higher cell densities. Densitometric measurements in this instance produced similar reflectance values at the homologous and most prominent heterologous reaction site when cell densities differed by approximately 3 and and pv. trunsluc.erzs 10 times (X. cunzpestris pv. ~vsicutoric~ antisera, respectively (Figs. 2a and 2b). In tests for optimization of the reaction, higher antibody concentration and (or) longer incubation in antisera, resulted in increasingly more intense cross-reactions among all the X. c-umpestris pathovars with both sera. Diluting the antisera and reducing the length of incubation in them, on the other hand, improved the specificity of the reactions. Considerable flexibility was found to exist as to the optimal antibody concentration and the length of incubation used. For instance, incubation

LAZAROVITS ET AL

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FIG. 2. Densitometric determinations of dot-ELISA reactions of Xanthomoncis cvlmpestris pv. trun.sluscens (X.t.) and pv. vesicatoria (X.V.) with antiserum produced with pv. vesicatoria (a) and pv. translu.scen,s ( h )and two conjugates, protein A -alkaline phosphatase (Prot. A - Alk. Phos.) and streptavidin-biotinylated peroxidase complex (Strept. perox.). Readings were obtained directly from photograph shown in Fig. 1. Ag, antigen; As, antiserum.

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FIG. 3. The effect of drop volume on detection by dot-ELISA of bacteria released from single lesions on pepper leaves harvested 7 d after inoculation with Xanthomonas campestris pv. vesicutoriu. The membrane was incubated in antiserum prepared against X . campestris pv. vesicatoria. Alkaline phosphatase conjugated protein A was used for detection. The apparent reaction seen with the control sample (H) is due to green pigment rather than enzymatic activiy.

times as short as 1 min, at a wide range of antibody concentrations, produced results comparable with that seen in Fig. 1, plates c and d. Neither the X. campestris pv. vesicutoriu or pv. truns1ucen.s antiserum reacted with the three non Xanthomonas sp. shown (Fig. 1, plates a-6).

Detection of bacteria from infected plant tissues Samples removed from drops of water, which contained pieces of plant tissue with a single bacterial lesion, showed a positive reaction with antiserum produced against X. campestris pv. vesicatoria. Undiluted samples reacted with similar intensity regardless of drop volume, but with dilutions of 1:5 and 1: 25 (Fig. 3), a decreasing gradient in reaction intensity was observed with increasing drop size. Quantitation, although not shown, confirmed this observation. At the 1: 125 dilution, only the sample from the 25-pL drop showed a reaction (Fig. 3). No reaction was observed when antiserum produced with X. campestris pv. translucens was used. Samples derived from healthy tissues also did not react with either antiserum (Fig. 3). Homogenized extracts of infected leaf tissues reacted positively at dilutions as low as 1:25 with X. campestris pv. vesicatoria antiserum (Fig. 4, membrane b) but not with X. campestris pv. translucens antiserum (Fig. 4, membranea). Healthy tissue extracts did not react with either serum (Fig. 4, membranes u and h ) . Cell-free extracts produced by boiling bacterial cells (Fig. 5) or infected tissues (Fig. 4, membranes c-e) contained antigenic components which reacted positively only to the homologous antiserum. The reactions seen with X. campestris pv. vesicatoria boiled extract (Fig. 5) were similar to that seen with intact cells (Fig. 1, plate u). With boiled extract from diseased tissues, the intensity of the reaction at various dilutions (Fig. 4, membrane c) was comparable with that seen with extracts produced by homogenization (Fig. 4, membrane b). Boiled extracts, however, produced more desirable results as they did not contain coloured pigments that could interfere when quantitative measurements were made. The reaction was

CAN. J. MICROBIOL. VOL. 33, 1987

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Sera Used FIG. 4. Detection of bacteria from infected tomato leaf tissue 7 d after inoculation with Xanthomonas cumpestris pv. vesicatoria. Samples Dl-D4 were harvested from infected sites and sample H from a healthy site. Membranes a and b were spotted with tissue homogenates and membrane c was spotted with cell-free extract obtained after boiling the lesions. Membrane a was incubated in antiserum produced against Xanthomonus campestris pv. trans1uscen.s (X.t.) and b and c in antiserum against pv. \,r,sic~aror.ia (X.V.). All were then reacted with alkaline phosphatase conjugated protein A. The apparent reactions seen with extracts D I to D4 and X. t . serum with the healthy tissue extracts (H) and both sera are due to pigments in the homogenates. Cell suspensions (2.5 x 10' cfu applied) and boiled cell-free extracts ( from lo7 cfu/mL) were included as controls for dot-ELISA. Key to strains applied: A. X. c~amprsrri.~ pv. \~c..cii.atoria;B . pv. pelmrgonium; C , pv. begonia; D, pv. translucens; E, pv. campestris.

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FIG. 5. The reaction of boiled cell-free extracts ( 10' cfu/mL) of five Xanthomonus campestris pathovars and three unrelated bacteria genera with antiserum produced against X. carnpestri.~pv . \,c.sic.ntoriu. Alkaline phosphatase conjugated protein A was used for detection. For key to bacteria strains, refer to Fig. 1.

monitored in all diagnostic tests by including membranes which had been spotted with living cells (2.5 x lo4 cells/spot) as well as boiled cell-free extracts of the five X. campestris pathovars (Fig. 4, membranes d and e).

Discussion Dot-ELISA on nitrocellulose membranes was found to be a simple, rapid, and sensitive assay for immunological characterization of phytopathogenic bacteria obtained either from cultures or diseased tissues. This is in agreement with results published by Malin et al. (1985) on the use of dot-EL1SA for identification of X. campestris pv. phaseoli. We have used this assay successfully to confirm of the presence of Erwinia amylovoru in pear tissues with suspected fire blight symptoms (Zutra et al. 1986), as well as for the detection of X. c-umpestris pathovars described here. Based on our experience, we predict similar success for tests with other bacterial genera. For qualitative determinations, no sophisticated equipment is required. It is possible, however, to adapt dot-ELISA into a sensitive, quantitative solid-phase immunoassay with the use of a reflectance densitometer (Towbin and Gordon 1984). For most

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LAZAROVJTS ET A L .

purposes, we found that equally accurate conclusions could be derived from visual inspection .The reactions with both conjugates showed a linear increase in intensity with increasing antigen concentration. With the homologous reaction this was generally found to occur in the range of 1600 to 25000 cfu. In most tests approximately 1600 to 6000 cfu per spot (ca. 0.3 cm2)provided the colour intensity that best differentiated homologous and heterologous reactions. The sensitivity obtained was ini'luenced to a limited extent by the antibody concentration and to a much greater extent by the nature of the conjugate used. Conjugated protein A was somewhat less sensitive than the streptavidin - biotinylated peroxidase reaction and perhaps because of this cross-reactions were less apparent with this conjugate. Overall, this technique provides comparable sensitivity to that obtained by immunofluorescence (Kishinevski and Bar-Joseph 1978; H. Y. Domen and A. M. Alvarez. 1979. Proc. Int. Conf. Plant Pathog. Bact. 4th. pp. 301-305) and by direct (H. Vruggink. 1978. Proc. Int. Conf. Plant Pathog. Bact. 4th. pp. 307-310) and indirect (M. Cambra and M . M . Lopez. 1978. Proc . Inf. Conf. Plant Pathog. Bact., 4th. pp. 327-331) ELISA performed in microtitre plates. A possible limitation of the dot-ELISA technique is the relatively small volume ( 1-5 kL) of sample that can be applied to the membrane. At present samples are required to contain populations in excess of lo6 cfu/mL in order to be within the limits of detection. Although such concentrations of cells can be obtained readily from cultures and diseased tissue, as illustrated in this study, they would not be expected to exist under more natural environments. Recently, however, several manifolds which permit the application of much larger volumes (millilitre quantities) of liquid have become commercially available. These devices should greatly increase the possible usefulness of dot-ELISA for screening samples with lower bacterial cell densities. Cross-reactions among all the pathovars of X. campestris could be demonstrated for both antisera when the streptavidin biotinylated peroxidase conjugate was used. The conflicts in the literature among those who obtained antisera with highly specific reactivities toward a single X. campestris pathovar (Schaad 1976, 1978; Starr 1981) and those that report that these pathovars have too many immunological similarities to be

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differentiated (Charudattan 1973; Starr 1981) may be related to differences in assay procedures. Certainly the availability of conjugates that allow one to manipulate both the degree of specificity and sensitivity will be a useful feature of dot-ELISA when used as a plant diseases diagnosis tool. It should, in any case, simplify the task of testing and comparing a large number of bacterial isolates with diverse preparations of antisera. BANTTARI,E. E., and P. H. GOODWIN. 1985. Detection of potato viruses S, X and Y by enzyme-linked immunosorbent assay on nitrocellulose membranes (Dot-ELISA). Plant Dis. 69: 202-205. CHARUDATTAN, R., R. E. STALL, and D. L. BATCHELOR.1973. Serotypes of X~lnthomonasvesicatoria unrelated to its pathotypes. Phytopathology, 63: 1260- 1265. CLARK, M. F., and M. BAR-JOSEPH. 1984. Enzyme immunosorbent assays in plant virology. In Methods in virology. Vol. 7. Edited by K. Maramorosch and H. Koprowski. Academic Press, New York. pp. 51-85. HAWKES,R., E. NIDAY,and J. GORDON. 1982. A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 11: 142- 147. KISHINEVSKI,B., and M. BAR-JOSEPH. 1978. Rhizobium strain identification in Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA). Can. J. Microbiol. 24: 1537- 1543. MALIN, E., E. L. BELDEN,and D. ROTH.1985. Evaluation of the radioimmunoassay, indirect enzyme-linked imminosorbent assay and dot blot assay for the identification of Xanthomonas campestris pv. phaseoli. Can. J. Plant Pathol. 7: 217-222. SCHAAD, N . W . 1976. Immunological comparison and characterization of ribosomes of Xanthomonas vesicatoria. Phytopathology ,66: 770-776. 1978. Use of direct and indirect immunofluorescence tests for identification of Xanthomonas campestris. Phytopathology, 68: 249-252. STARR, M. P. 1981. The genus Xanrhomonas. In The prokaryotes. A handbook on habitats, isolation and identification of bacteria. Vol. 1 . Edited by M. P. Starr, H . Stolp, H . G. Truper, A. Balows, and H. G. Schelgel. Springer-Verlag, Berlin. pp. 742-763. TOWR~N, H . , and J . GORDON. 1984. Immunoblotting and dot immunobinding-current status and outlook. J. Immunol. Methods, 72: 313-340. ZUTRA, D., E. SHABI, and G. LAZAROVITS. 1986. Fire blight on pear, a new disease in Israel. Plant Dis. 69: 107 1- 1073.