DETECTION AND IDENTIFICATION OF Ralstonia [PDF]

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Indonesian Journal of Agriculture 1(1), 2008: 13-21 ... Detection and identification of Ralstonia solanacearum

DETECTION AND IDENTIFICATION OF Ralstonia solanacearum STRAINS USING THE INDIRECT ELISA TECHNIQUE1)

M. Machmud and Yadi Suryadi Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development Jalan Tentara Pelajar No. 3A, Bogor 16111

ABSTRACT Several techniques for early and rapid detections of Ralstonia solanacearum have been developed as components in the integrated control of bacterial wilt. The DNA based techniques are highly effective in detecting the bacterium, but they require sophisticated and expensive materials and impractical for field applications. The Enzyme-Linked Immunosorbent Assay (ELISA) is one of the serological techniques that is effective for detection and identification of bacterial plant pathogens, because it is relatively rapid, inexpensive, does not require sophisticated equipment, and applicable under field conditions. Modifications have been made by researchers to improve sensitivities of the detection, including those for R. solanacearum, and among them is the Indirect ELISA technique. A laboratory study was done to produce polyclonal antibody (PAb) to R. solanacearum and to apply the antibody for detection of R. solanacearum strains representing different hosts, races, and biovars using the Indirect ELISA technique. The results showed that PAb to R. solanacearum was producible on white rabbits using three different immunization schemes at titers ranging from 128 to 4096. The Indirect ELISA technique using the PAb was applicable for detection of R. solanacearum strains representing race 1 biovar 3, race 2 biovar 1, and race 3 biovar 2, either from pure cultures, soils or plant parts. The lowest detection level of the ELISA technique was 10 3 cells/ml. [Keywords: Ralstonia solanacearum, detection and identification, indirect ELISA, bacterial wilt control]

INTRODUCTION Bacterial wilt caused by Ralstonia solanacearum (Pseudomonas solanacearum) is one of the most important diseases on agricultural crops, particularly on food and horticultural crops. Yield losses due to the disease on the different crops varied and difficult to measure accurately, but were estimated at 15-35% on tomato, 35-60% on potato, and 30-65% on groundnut (Hayward 1994; CAB

1)

Article in bahasa Indonesia has been published in Penelitian Pertanian Tanaman Pangan Vol. 25 No. 2, 2005 p. 91-99.

International 2004). In the last decade, banana plants in various Indonesian regions were seriously damaged by the disease that caused farmers suffered billion rupiah due to yield losses annually (Sulyo 1992). Efforts have been done to control the disease using one or more of the integrated control components. The results however have not been encouraging (Robinson-Smith 1993). The disease is difficult to control, particularly because the pathogen is capable of surviving and adapting to its ecosystem (Hayward 1994). R. solanacearum is widely distributed in various regions of the world, particularly in the tropical and subtropical regions, from lowland to highland area over 2500 m asl. The pathogen has a wide host range, over 400 plant species representing over 80 families. Strains of R. solanacearum have been grouped into five races (races 1 to 5) based on their host ranges, and five biovars (biovars I to V) based on their nutritional requirements, particularly carbohydrates and organic acids (Seal and Elphinstone 1994). The pathogen is also capable of surviving in the seeds, soils, and rhizospheres of nonhost plants for quite a long time (Janse 1988; Seal and Elphinstone 1994). Early disease diagnoses are important steps for a successful disease control. The first important step in the disease diagnoses is accurate detection of the pathogen. This needs to be done using an effective and efficient technique that enables to control the disease effectively, quickly, and accurately. An effective and efficient detection should fulfill five criteria, i.e. quick, sensitive, accurate, applicable in the field, and economical (cheap) (Seal 1994 and Elphinstone). Enzyme-Linked Immunosorbent Assay (ELISA) is a serological technique that meets those requirements. It is a very popular and promising technique for diagnosis of plant diseases, particularly for those caused by viruses and bacteria (Converse and Martin 1990). This technique has been used by many users in Indonesia for detection of plant diseases (Machmud et al. 1996). Presently, however, ELISA kits as well its components still have to be imported with an expensive price; it took Rp15,000-Rp20,000 to test a plant sample

14

M. Machmud and Yadi Suryadi

(Agdia Inc., Leckhart, Indiana). This price can be reduced when the kits are produced domestically. The basic technique of ELISA is serology, the reaction between antigen (Ag) and antibody (Ab). This technique, however, needs certain enzymes and substrates to label the reaction that enable to produce color that readable by naked eyes or using an electronic tool, ELISA Reader (Converse and Martin 1990; McLaughlin and Chen 1990). The original ELISA technique has been modified to improve its effectiveness and given different names, such as Double Antibody Sandwiched-ELISA (DAS-ELISA), Direct ELISA, Indirect ELISA, and Dot Blot ELISA (Canale et al. 1983; Stobbs and Barker 1985; Yadi et al. 1998). These techniques have not been evaluated for their effectiveness to detect R. solanacearum, either from pure cultures, plants or soils. Since 1995, efforts to adopt and modify the ELISA technique have been done in the Research Institute for Food Crops Biotechnology, Bogor, by improving techniques for production of policlonal antibodies (PAbs) and components of ELISA kits for some viral and bacterial plant pathogens including R. solanacearum (Machmud et al.. 1996, 1997, 1998, 1999). The goal of the research is to obtain effective and efficient techniques for production of PAbs and to assemble ELISA kits, so that it becomes cheaper. This research was aimed to: (1) produce PAb to R. solanacearum, (2) test the effectiveness of three ELISA techniques to detect and identify R. solanacearum, and (3) test the sensitivity of ELISA technique to detect R. solanacearum from plants and soils.

MATERIALS AND METHODS This research was conducted in the laboratory of the Indonesian Center for Biotechnology and Genetic Resources Research and Development, Bogor, West Java from June to October 2004. The research consisted of three trials, i.e. (1) production of PAb for R. solanacearum, (2) evaluating the effectiveness of three ELISA techniques for detection and identification of R. solanacearum, and (3) testing the effectiveness of the Indirect ELISA technique for detection of R. solanacearum from plants and soils.

Production of PAb for R. solanacearum Productions of the PAb for R. solanacearum were done in 5-6 month-old hybrids of the New Zealand white rabbits. Two isolates of R. solanacearum were used for sources of antigen (Ag), i.e. Ps 9601 representing the race 1 biovar 3, which was isolated from groundnut plants in Muara Experimental Farm, Bogor, West Java, and Ps 2002-09

representing the race 3 biovar 2, which was isolated from potato plants in Margahayu village, Lembang, West Java. Each of the isolates was cultured on the sucrose peptone agar (SPA) plates (Machmud et al. 1996). Bacterial suspensions were prepared by suspending 48 hour-old culture of each isolate in phosphate-buffered saline (PBS) solutions of 0.1 M, pH 7.2; the density of the bacterial suspension was measured at 1010 cells/ml using a Hitachi U2000 spectrophotometer. There were 15 different treatment combinations used in the PAb production, these were combinations of Ags for R. solanacearum and five rabbit immunization techniques. The three different Ags for R. solanacearum were: (1) formalin-killed R. solanacearum cells (Ag1); (2) glutaraldehyde-killed cells (Ag2), and (3) heat-killed cells (Ag3). The Ag preparations followed the technique of Machmud et al. (1996). The five immunization techniques were: (1) intramuscular injection (IM); (2) intraperitoneal injection (IP); (3) intravenal injection (IV); (4) a combination of IV and IP (IV + IP), and (5) standard immunization of Robinson-Smith (1993) as a control. The IM, IP, dan IV techniques used in this study followed those of Ball et al. (1990), while the combination of IV+IP followed that of Machmud et al. (1996). Each immunization technique was done on two rabbits as replicates. Bleeding and antiserum collection, purification of PAb, and measurements of PAb titers were done following the tehnique of Ball et al. (1990). PAb for R. solanacearum race 1 biovar 3 (isolate PS1006) was coded PAb1 and that for race 3 biovar 2 was coded PAb2. Testing the antibody reaction specificities of the PAb1 and PAb2 to strains of R. solanacearum were done using the Indirect ELISA technique of Robinson as modified by Machmud et al. (1996). The PAb specificities were tested on nine isolates of R. solanacearum representing different races and biovars, one bacterial species genetically close to R. solanacearum (Pseudomonas syzygii) and two other bacterial plant pathogens (P. syringae pv. glycinea and Xanthomonas axonopodis pv. glycines). Results of the ELISA testing were examined visually based on color changes of the substrate in wells of the ELISA plate. The reaction was assessed positive or specific if the reaction between PAb and Ag give a positive reaction as marked with a color change of the substrate becomes blue. If the substrate color does not change or remain yellow, hence PAb does not react with Ag and reaction is expressed negative. Each examination used two replications.

Sensitivities of ELISA Techniques for the Detection of R. solanacearum Three ELISA techniques were tested for their sensitivities to detect R. solanacearum that is: (1) the Direct ELISA

15

Detection and identification of Ralstonia solanacearum ...

technique of Robinson-Smith (1993), (2) the Indirect ELISA technique according to Machmud et al. (1996), and (3) the Nitrocellulose Membrane-ELISA (NCM-ELISA) technique of Yadi et al. (1998). The trial was done using PAb1 and Ag2. The original Ag2 was prepared by suspending 48 hour-old culture of R. solanacearum Ps9601 in sterile 0.1 M PBS pH 7.2 to a concentration of approximately 1010 cells/ml. At the time of examination, a serial dilution of the Ag was made to concentrations of 108, 106, 105, 104, and 103 cells/ml. Each treatment was replicated five times. Sensitivities of the techniques were observed visually or using a Thermo Lab System Opsys MR ELISA reader at 405 nm wavelength (OD405). Visual observations were based on color changes of the substrate; a blue color indicates a positive reaction. The most sensitive technique is that produces positive reaction at the lowest Ag concentration.

Specificities of R. solanacearum PAb Using the Indirect ELISA Technique This activity was conducted to test specificities of PAb1 and PAb2 reactions against Ag of R. solanacearum strains and other pathogens, i.e., 10 isolates of R. solanacearum representing different strains, one bacterial pathogen closely related to R. solanacearum (P. syzygii), and two other bacterial pathogens (P. syringae pv. glycinea and X. axonopodis pv. glycines). The testing was done using the Indirect ELISA technique of Machmud et al. (1996). Results of the examination were observed visually or using a Thermo Lab System Opsys MR ELISA reader at 405 nm wavelength. Visual observations were based on color changes of the substrate; a blue color indicates a positive reaction. Each examination was replicated twice.

Sensitivities of the Indirect ELISA Technique for the Detection of R. solanacearum from Plants and Soils Testing the Indirect ELISA technique sensitivity was done in two ways: (1) using artificially inoculated soil and plant samples, and (2) using naturally infested soil and plant samples. PAb1 was used as the reactant antibody and bacterial suspension of a 48 hour-old R. solanacearum Ps culture was used as a positive control.

Testing of artificially infested plants and soils Prior to the testing, sterile soils and groundnut plants were prepared. Soil samples collected from Cikeumeuh, Bogor,

were sterilized by autoclaving at 121oC for 30 minutes. One month-old groundnut plants of Kelinci cultivar were prepared in six polybags, each polybag contained 500 g of sterile soils. The polybags were then infested evenly with suspensions of R. solanacearum PS9601 at the rate of 108, 106, 105, 104, and 103 cells/g soils, respectively. Hereinafter, 100 g soil samples were taken from each polybag and placed in a 250 l Erlenmeyer flasks containing 100 ml 0.1M PBS pH 7.2. Ten ml of mixtures were taken from each flask, centrifuged at the rate of 1000 rpm for 10 minutes, and supernatants were collected for use as Ags. This was a combination of techniques used by Janse (1988) and Seal et al. (1992) . Plant samples were prepared from six one-month old groundnut plants grown in the sterile soil. Each plant was ground for 5 minutes using a blender containing 100 ml 0.1 M PBS buffer. The plant extracts were distributed equally into six 250-ml Erlenmeyer flasks. Ten ml of sterile plant extract was taken from each flask and placed in a sterile tube. Into each of the Erlenmeyer flasks containing the plant extracts, R. solanacearum suspension that has been prepared earlier was added to make a serial dilution of 108, 106, 105, 104, and 103 cells/ml. Subsequently, 10 ml of the plant extract was taken from each inoculum’s dilution and placed in a centrifuge tube and centrifuged using a benchtop centrifuge at 1000 rpm for 10 minutes. The supernatants were resuspended in 0.1 M PBS pH 7.2 and used as sources of Ags. The soil and plant extracts were used as sources of Ags, while PAb1 and PAb2 were used as the Abs for testing the sensitivity of the Indirect ELISA. Sensitivity of the technique was evaluated based on the results of visual observations and using an ELISA Reader Thermo Lab System Opsys MR.

Testing on plant and soil samples from the fields Soil and plant samples used in this test were collected directly from the field. These samples consisted of: (1) soil of groundnut rhizosphere; (2) soil of potato rhizosphere, (3) soil of tomato rhizosphere, (4) groundnut seeds infected with R. solanacearum, (5) potato tubers infected with R. solanacearum, (6) groundnut plants infected with R. solanacearum, (7) potato plants infected with R. solanacearum, (8) tomato plants infected with R. solanacearum, (9) healthy groundnut seeds (negative control), (10) healthy groundnut plants (negative control), (11) healthy potato plants (negative control), (12) healthy tomato plants, (13) healthy potato tubers (negative control), (14) pure culture of R. solanacearum Ps 9601 (positive

16

M. Machmud and Yadi Suryadi

control), (15) pure culture of R. solanacearum Ps 2002-09 (positive control), and (16) 0.1 M PBS pH 7.2 (negative control). Extracts of the soil and plant samples were prepared as mentioned earlier. The soil and plant extracts were used as sources of Ags to test the sensitivity of the ELISA technique, while PAb1 and PAb2 were used as the antibodies. Sensitivity of the technique was evaluated based on the results of visual observations and using an ELISA Reader Thermo Lab System Opsys MR.

RESULTS AND DISCUSSION Production of Polyclonal Antibody Immunization of New Zealand white rabbit hybrids with 24 treatment combinations involving two isolates of R. solanacearum (Ps 9601 and Ps2002-09), three different Ags (Ag1, Ag2, and Ag3), and four immunization techniques (IM, IP, IV, and IV+IM), all produced PAbs with titers ranging from 256 to 4096. Titer is the reversed value of the smallest dilution PAb that still gives a positive reaction with the Ag. The positive reaction of PAb-Ag is indicated by the formation of microagglutinate using the slide microagglutination technique as shown in Figure 1 (Ball et al. 1990). Rabbit immunization by the different Ags and using the different techniques produced PAbs with varied titers

(Table 1). The antibody titers were not influenced by the R. solanacearum strains used as the Ag sources, but more affected by the form of antigens and the rabbit immunization techniques. The titer of PAb produced using R. solanacearum Ps 9601 representing race 1 biovar 3 was similar to that produced using isolate Ps 2002-09 representing race 3 biovar 2, which were ranging from 128 to 4096. The type of Ag used for immunization significantly influenced the titer of PAb produced. Rabbit immunizations using formalinized bacterial cells (Ag1) or glutaraldehydekilled bacterial cells (Ag2) produced PAbs with titers higher than that immunized using the heat-killed bacterial cells (Ag3), which were 2048, 4096, and 1024 of PAb1, and 4096, 4096, and 1024 of PAb2 (Table 1). The immunization techniques used in the PAb production also influenced the PAb titers. The IV technique and a combination of IV + IP produced the highest titers of PAb (4096), i.e. PAb2 using Ag2 and PAb1 using Ag1. The IP technique produced the lowest PAb titer (256) than the others, either using Ag1 or Ag2. The IM technique and the Robinson-Smith technique (control ) produced PAbs with titers only 512. In the earlier study, Machmud et al. (1996; 1999) reported that rabbit immunizations by a combination of IV and IP and heat-killed Ag or glutaraldehyde-killed Ag produced PAbs with titers ranging from 512 to 1024. In 1993, Robinson-Smith produced PAbs of different strains of R. solanacearum using the IM immunization with titers ranging from 8 to 512. Ball et al. (1990) reported that a

Figure 1. Reaction between polyclonal antibody (PAb) and antigen (Ag) of Ralstonia solanacearum based on the microagglutination technique on a microscope glass slide; above = negative reactions, below = positive reactions, in the form of agglutinant or coagulant.

17

Detection and identification of Ralstonia solanacearum ...

Table 1. Titers and specificities of Ralstonia solanacearum polyclonal antibodies produced in rabbits using five different immunization techniques. Titer of PAb Immunization technique

Intravenal (IV) Intraperitoneal (IP) Intramuscular (IM) Combination of IV dan IP Control Average

PAb1

PAb2

Average

Ag1

Ag2

Ag3

Ag1

Ag2

Ag3

2048 128 256 2048 512 1076

4096 256 512 4096 512 1946

1024 256 128 1024 128 512

2048 128 256 4096 512 1485

4096 256 512 4096 512 1946

1024 128 256 1024 128 538

2806 268 320 2730 384

Ag1 = formalinized antigen using 2.5% formaldehyde; Ag2 = glutaraldehyde-fixed antigen using 2.5% glutaraldehyde, and Ag3 = heatkilled antigen by heating in boiling water (100oC) for 2 hours. Control = the technique of Robinson-Smith (1993). Titer of PAb is the inversed/reversed value of the smallest PAb dilution that still give a positive reaction based on the microagglutination technique of Ball et al. (1990).

Table 2. Specificities of two PAbs of R. solanacearum for detection and identification of different strains of R. solanacearum. Antibody Isolat

Ps 80-09 Ps 9510 Ps 9601 Ps 9602 Ps 2002-05 Ps 2002-07 Ps 2002-01 Ps 2002-04 Ps 2002-12 Ps 2002-19 P. sysygii Psg 01-02 Xcg 01-01 Control

Host plant

Ginger, Curup, Bengkulu Groundnut, Kalijati, Subang Groundnut, Cikeumeuh, Bogor Groundnut, Muara, Bogor Tomato, Cipanas, Cianjur Chili, Cikole, Lembang Banana cv. ambon, Cikeumeuh, Bogor Banana cv. kepok, Loji, Bogor Potato, Pangalengan, Bandung Potato, Margahayu, Lembang Clove, Pamoyanan, Bogor Soybean, Cikeumeuh, Bogor Soybean, Cikeumeuh, Bogor Phosphate buferred saline (PBS)

Strain

PAb1 Ag2

PAb2 Ag2

Ras

Biovar

Reaction

OD405

Reaction

OD405

1 1 1 1 1 1 2 2 3 3

2 3 3 3 3 3 1 1 2 2

+ + + + + + + + + + + -

0.822 0.868 0.986 0.930 0.678 0.758 0.789 0.806 0.848 0.892 0.582 0.092 0.113 0.102

+ + + + + + + + + + + -

0.788 0.786 0.827 0.824 0.906 0.890 0.782 0.826 0.942 0.886 0.490 0.124 0.106 0.082

Value of OD405 = average of two replications; standard deviation = 0.122. A405 = Asorbance value measured using ELISA Reader Thermo Lab System Opsys MR in the 405 nm wavelenght. + = positive reaction; - = negative reaction. Ps = R. solanacearum; P. syzygii = pathogen genetically close to R. solanacearum; Psg = P. syringae pv. glycinea; Xcg = Xanthomonas axonopodis pv. glycines.

good rabbit immunization technique produces PAbs with titers > 512. The IV immunization and a combination of IV + IP are two best techniques for production of PAb for R. solanacearum. The best Ags to use in the rabbit immunizations were Ag1 and Ag2, the formalin-killed cells and glutaral dehyde-killed cells of R. solanacearum, respectively.

Specificity of PAb for R. solanacearum Using the Indirect ELISA The PAbs produced using Ags of two different strains of R. solanacearum representing the race 1 biovar 3 and race 3 biovar 2 (Ps 9601 and Ps 2002-09) showed specific reactions to R. solanaceaum, but showing a slight cross

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M. Machmud and Yadi Suryadi

substrates and optical density value at 405 nm wavelength (OD405) using the ELISA Reader. This indicates that the Indirect ELISA technique was one level more sensitive than the other techniques. Robinson-Smith (1993) and Machmud et al. (1996; 1999) reported similar results when comparing sensitivities of the techniques using other R. solanacearum strains and other pathogens. McLaughlin and Chen (1990) also prefer to use the Indirect ELISA technique for detection of bacterial pathogen. They reported that besides its sensitivity, the technique also uses a secondary Ab-conjugate that available commercially, thus enable to shorten the time of testing, without having to prepare a primary Ab-conjugate, since it is time consuming and needs a special skill.

reaction with genetically close related species, such as P. syzygii. However, PAbs were not reacted positively with Ags of other bacterial species (Table 2). PAb1 that was produced using Ag Ps 9601, representing the race 1 biovar 3, and PAb2 that was produced using Ag Ps2002-09, representing the race 3 biovar 2, each reacted positively with 10 R. solanacearum isolates representing race 1 biovar 3, race 2 biovar 1, and race 3 biovar 2 from different host ranges and localities. PAb of R. solanacearum also reacted positively with Ag of P. syzygii, the xylem-limited bacterial pathogen of clove tree that has a close genetic relationship with R. solanacearum. This result indicates that PAb for R. solanacearum does not react specifically to certain R. solanacearum strain, even reacts with other patogen which has a close genetic relationships. To improve specificity of the antibodies, Robinson-Smith (1993) produced monoclonal antibodies that gave a specific strain reactions. The two PAbs did not react with P. syzygii pv. glycinea Psg 01-02, the bacterial blight pathogen of soybean, and X. axonopodis pv. glycines Xcg 01-01, the bacterial pustule pathogen of soybean. A similar results were reported by Robinson-Smith (1993) when testing specificities of PAb of different strains of R. solanacearum using the same technique.

Testing Sensitivity of Indirect ELISA for Detection of R. solanacearum from Soils and Plants

Testing using artificially inoculated soil and plant samples The Indirect ELISA technique was used in the detection of R. solanacearum from artificially inoculated soil and plant samples (Table 4). Based on visual observations and using the ELISA Reader, sensitivity of the technique to detect R. solanacearum from plant extracts was 104 cells/ml, one level higher than its detection sensitivity for R. solanacearum from suspension of pure culture (103 cells/ ml), but one level lower than its detection sensitivity from soil extract (105 cells/ml). These results were in accordance with those of Robinson-Smith (1993) and Machmud et al. (1996). The detection limit of R. solanacearum from soil

Effectiveness of Three ELISA Techniques Results of testing on the effectiveness of the three ELISA techniques, i.e. Direct ELISA, Indirect ELISA, and NCMELISA, showed different effectiveness of detection (Table 3). The Indirect ELISA technique was capable of detecting R. solanacearum of 103 cells/ml, while the Direct ELISA and the NCM-ELISA techniques detected only up to 104 cells/ml. This was indicated based on color changes in the

Table 3. Sensitivity of three modified ELISA techniques based on visual observation results and absorbant value, Bogor, 2004. Reaction on antigen density (cells/ml)

ELISA techniques 10 Indirect ELISA Direct ELISA NCM-ELISA

8

+++ (0.924) +++ (0.852) +++

10

7

++ (0.726) ++ (0.706) +++

10

6

++ (0.682) ++ (0.663)

10

5

++ (0.622) + (0.462) ++

10

4

+ (0.456) (0.209) +

10

3

+ (286) (0.205) -

Negative control (0.126) (0.135) -

+ = positive and - = negative reaction based on visual observation. Numbers in parentheses are optical density measured using ELISA Reader Thermo Lab System Opsys MR in the 405 nm (OD405) wavelength; standar deviation = 0.132. NCM-ELISA results can only be read visually.

19

Detection and identification of Ralstonia solanacearum ...

Table 4. Sensitivity of the Indirect ELISA technique to detect Ralstonia solanacearum in soils and plants from the fields, Bogor, 2004. Antigen content (cells/ml)

Source of antigen

Pure bacterium R. solanacearum Groundnut var. Kelinci Sterilized soil from Cikeumeuh, Bogor

Control 10 8

10 7

10 6

10 5

10 4

10 3

+++ (0.924) +++ (0.858) +++ (0.812)

++ (0.728) ++ (0.682) +++ (0.628)

++ (0.682) ++ (0.604)

++ (0.602) ++ (0.542) ++ (0.492)

+ (0.556) + (0.425) + (0.280)

+ (0.386) (0.305) (0.168)

(0.582)

(0.176) (0.132) (0.126)

Soil and extract of groundnut plant diluted with R. solanacearum suspension at different cell intensities. + = positive reaction and - = negative reaction based on visual observation. Numbers in parentheses are optic density measured using ELISA Reader Thermo Lab System Opsys MR in the 405 nm (OD405) wavelength; standard deviation = 0.156.

Table 5. Sensitivity of the Indirect ELISA technique for the detection of Ralstonia solanacearum (RS) from soil and plant samples collected from the fields, Bogor, 2004. ELISA reaction using Soil or plant sample

Soil of groundnut rhizosphere Soil of potato rhizosphere Soil of tomato rhizosphere Groundnut seeds infected with RS Potato tubers infected with RS Groundnut plants infected with RS Potato plants infected with RS Tomato plants infected with RS Healthy groundnut seeds (negative control) Healthy groundnut plants (negative control) Healthy potato plants (negative control) Healthy tomato plants Healthy potato tubers (negative control) RS pure culture of isolate Ps 9601 (positive control) RS pure culture of isolate Ps 2002-09 (positive control) Phosphate-buffered saline (PBS, negative control)

PAb1

Origin (locality)

Cikeumeuh, Bogor Cipanas, Cianjur Margahayu, Lembang Cikeumeuh, Bogor Cipanas, Cianjur Cikeumeuh, Bogor Cipanas, Cianjur Margahayu, Lembang Cikeumeuh, Bogor Cikeumeuh, Bogor Margahayu, Lembang Margahayu, Lembang Cipanas, Cianjur Race 1 Biovar 3 Race 3 Biovar 2

PAb2

Vis.

OD405

Vis.

OD405

+ + + + + + + + + + -

0.678 0.542 0.468 0.860 0.672 0.756 0.782 0.816 0.148 0.192 0.182 0.210 0.153 0.884 0.942 0.132

+ + + + + + + + + + -

0.620 0.586 0.568 0.840 0.778 0.784 0.828 0.860 0.184 0.160 0.156 0.202 0.138 0.926 0.896 0.168

Vis. = visual observation based on color changes of substrates; + = positive reaction, - = negative reaction. A405 = absorbance at 405 nm wavelength as measured using the Thermo Lab System Opsys MR ELISA reader with a standard deviation = 0.128.

samples was low, but it can be improved by modifying the components of the soil extraction by adding cholic acid and polyvinyl pirollydon (PVP) (McLaughlin et al. 1989). According to Priou (1997) in Yadi et al. (1998), improvement of detection limit can also be done using the enrichment technique, by preculturing extract of suspected plants containing R. solanacearum in an enrichment medium.

Testing of plants and soil samples collected from the fields Results of testing the sensitivity of the Indirect ELISA technique using naturally infested soil and plant material collected from infested fields showed similar results to that using the artificially infested soils and plant materials (Table

20

M. Machmud and Yadi Suryadi

5). The technique was applicable using the PAb1 and PAb2, to detect R. solanacearum from soil of the groundnut rhizospheres, potato rhizospheres, and rhizospheres of tomato plants showing bacterial wilt symptoms. The technique was also able to detect R. solanacearum directly from the groundnut seeds, potato tubers, as well as from groundnut and potato plants. The healthy plant samples of groundnut, potato, and tomato did not show positive reactions. The high values of OD405 (0.672-0.680) on both seed and plant samples indicated that populations of R. solanacearum in the plant samples were high. This was indicated with the OD405 values of the positive control, which was using a suspension of bacterial culture at a concentration of 3 x 108 cells/ml. The OD405 values in the soil samples from groundnut, potato, and tomato rhizospheres were lower than those on seed or plant samples (0.468-0.678). These may happen due to two posssibilities, either the R. solanacearum populations in the rhizospheres were lower than those in the plant samples or sensitivities of the Indirect ELISA technique for the detection of R. solanacearum in the soil was not optimal due to the presence of soil inhibitor. The chemical components of the soil inhibitor have not been fully identified; some of them, however, were phenolic compounds (Stobbs and Barker 1985; Janse 1988; Seal et al. 1992).

CONCLUSION AND SUGGESTION

R. solanacearum PAbs were successfully produced in hybrids of New Zealand white rabbit with titers ranging from 128 to 4096. Rabbit immunization using IV technique or a combination between IV and IP gave the best result. The Indirect ELISA technique was capable of detecting R. solanacearum of up to 103 cells/ml, more sensitive than either the Direct ELISA or the NCM ELISA technique. The Indirect ELISA technique was applicable to detect and identify R. solanacearum directly from the plants and the soils. A mass production system for PAb of R. solanacearum needs to be developed for commercialization and practical application purposes in the field. Indirect ELISA kits and the protocol may be made for its users in the field for different purposes, such as study on ecology of R. solanacearum as well as epidemiology of the bacterial wilt, seed and plant health testing for plant quarantine and certification purposes.

ACKNOWLEDGEMENT

The authors wish to thanks Ms. Endang Windiyati and Mr. Wawan for their technical supports in conducting the experiments.

REFERENCES

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