Abstract: The soft rot bacteria Pectobacterium and Dickeya species cause economically important diseases on potato crops. Specific and efficient detection methods are essential to investigate the ecology and pathogenesis of the soft rot bacteria as well as in seed certification programmes. Polyclonal antibodies against potato soft rot bacterium; Pectobacterium carotovorum subsp. carotovorum were generated in female New Zealand white rabbits. Determination of the optimum period to collect the antiserum (As) using indirect Enzyme-Linked Immunosorbent Assay (ELISA) showed that, the first collecting date 7 days after the last injection was the best followed by the second collecting date 14 days in both killed (As1) and live (As2) bacterial antisera. The best positive values of antiserum titer were obtained with As1 (7 days) up to dilution of 1:1.28×104 then As1 (14 days) 1:6.4×103 and As2 (7 or 14 days) 1:8×102. Efficiency of the antiserum to comparing among six isolates causing soft rot, Pectobacterium carotovorum subsp. carotovorum (PCCS63) which used for antisera production, P. carotovorum subsp. carotovorum (IPO1949), P. carotovorum subsp. atrosepticum (1007), P. carotovorum subsp. wasabiae (ipp041), Dickeya dianthicola (IPO2114) and Dickeya chrysanthemi (DSM4610) revealed that, our polyclonal antiserum had a high sensitivity to react with P. carotovorum subsp. carotovorum isolates PCCS63 and IPO1949 than with D. chrysanthemi DSM4610 isolate and the least coupling was found with P. carotovorum subsp. wasabiae ipp041 and Dickeya dianthicola IPO2114 isolates. The produced antiserum was more sensitive to detect P. carotovorum subsp. carotovorum isolates than the other soft rot bacteria.
INTRODUCTION
Contamination of seed tubers with soft rot bacteria is one of the biggest problems in seed potato production. These bacteria cause blackleg, rotting of potato stems in the field and soft rot of tubers during storage. Seed lots are discarded frequently due to blackleg in the field or soft rot in storage. Latent infections in seed tubers often result in blackleg appearing unpredictably in the field. Furthermore, these bacteria also cause severe storage losses for the potato processing industry, especially in large warehouses without refrigeration facilities. In temperate climates Pectobacterium carotovorum subsp. carotovorum (Pcc) (formerly, Erwinia carotovora subsp. carotovora; Gardan et al., 2003), has been regarded as the causal agent of soft rot (Perombelon, 2002; Toth et al., 2003) while Pectobacterium atrosepticum (formerly, Erwinia carotovora subsp. atroseptica; Gardan et al., 2003) has been typically considered to cause both blackleg in the field as well as soft rot during storage.
Dickeya spp. (formerly, E. chrysanthemi and P. chrysanthemi, Samson et al., 2005) have long been recognized as pathogens of ornamental plants particularly in tropical and subtropical climates. In warm climates, Dickeya has also been reported to cause both blackleg and soft rot of potato (Perombelon, 2002). Dickeya strains have been isolated repeatedly from potatoes during recent decades and the disease has rapidly spread in Western Europe during recent years and Africa (Palacio-Bielsa et al., 2006; Behiry, 2013). Variability within the former species Erwinia chrysanthemi is large and the strains were divided into several biovars and pathovars (Boccara et al., 1991; Nassar et al., 1996; Avrova et al., 2002). The variability was studied with different methods, such as PCR amplification and sequencing or RFLP of 16S or 16S-23S rDNA intergenic spacer (Toth et al., 2001; Fessehaie et al., 2002) and ELISAs (Alarcon et al., 1995; Duarte et al., 2004).
Enzyme Iinked Immuno Assay (ELISAs) is one of the most sensitive and frequently used methods for estimation of pathogens (i.e., bacteria) and Lipopolysaccharide (LPS) molecules. Thus it may be possible for ELISAs to detect low levels of soft rot Pectobacteria. However, sensitivity was limited to107 CFU mL–1, in ELISA compared to a sensitivity of 103 CFU mL–1 for a PCR test using published primers directed to the pectate lyase gene (Nassar et al., 1996). An enrichment ELISA procedure has been used in the Netherlands for routine detection of Dickeya spp. include cost efficiency and a correlation of 95% between this method and PCR testing (Van Der Wolf et al., 2006). Whole bacterial cells (i.e., Pcc) and Lipopolysaccharide (LPS), the major surface component of gram-negative bacteria specifically implicated in plant pathogenesis, are major antigens capable of initiating an immune response in various animal hosts. (Bassoriello, 2010) developed polyclonal ELISAs antibodies against P. carotovorum subsp. carotovorum Lipopolysaccharide (LPS) and Heat-killed (HK) or live P. carotovorum subsp. carotovorum (Pcc) limited the detection and quantification of the pathogen. The objectives of this work aimed to develop and characterize polyclonal rabbit serum raised against heat-killed P. carotovorum subsp. carotovorum and P. carotovorum. subsp. carotovorum LPS to determine which elicits a more specific response to P. carotovorum subsp. carotovorum; determine whether cross-reactivity exists among antibodies specific to P. carotovorum subsp. carotovorum and Pectobacterium spp. or other related genera that may be cause soft rot or blackleg of potato and develop a polyclonal rabbit serum-based ELISA for detection of P. carotovorum subsp. carotovorum and other soft rot pathogens.
MATERIALS AND METHODS
Antiserum production: Suspensions (ca. 108 CFU mL–1 in phosphate buffer saline, PBS) of heat killed, live cells from soft rot bacterial isolate P. carotovorum subsp. carotovorum PCCS63 previously identified by biochemical and molecular tests (Behiry, 2013) and lipopolysaccharides of P. carotovorum (Pcc LPS) and P. atrosepticum (Pca LPS) (provided by Dr. Mammdoh Ismail, Fac. of Agric., El-Menia, Egypt) were used to produced antisera in four female New Zealand white rabbits by five injections course, (0.5, 1.0, 1.5, 2.0 and 2.5 mL, respectively) the first one subcutaneous and the others in muscle thigh with 7 days interval (Al-Ani et al., 2004).
Bleeding from the marginal ear vein was carried out after 7 and 14 days from the last injection and left for clotting 2-3 h at room temperature then stored over night at 4°C. Antisera were clarified by centrifugation at 5000 rpm for 10 min and stored in the presence of 0.05% sodium azide at -20°C till used (Kayali et al., 2004). Normal serum was obtained from rabbit before immunization to use as a control in subsequent tests.
Indirect ELISA
Determination of the optimum antiserum collecting period: Indirect ELISA was carried out as described by Younes (1995). Bacterial suspension was prepared in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.2) at a concentration of ca. 108 CFU mL–1. Wells were coated by adding 100 μL of the bacterial suspension to the bottom of the well (2 wells per each antiserum dilution) and incubated over night at 4°C. The plates were rinsed three times with washing solution PBS -Tween 20 for 3 min each.
Eight dilutions of double fold up to 1:12800 for normal serum (as control) and the two antisera obtained after 7 and 14 days of the last injection in serum buffer (PBS-Tween 20 containing 2% soluble polyvinylpyrrolidone, 0.2% BSA) were used. One hundred microliter aliquots from the diluted normal serum and antisera were added to each well, after which the plates were incubated at 37°C for 2 h and then washed as before.
Goat anti-rabbit gamma globulin conjugated to alkaline phosphatase (Sigma No 2937) was diluted 1:20000 in serum buffer and 100 μL were added to each well and incubated at 37°C for one hour and then washed as before.
One hundred microliter of the enzyme substrate, 0.5 mg mL–1 para-nitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8 were added to each well and incubated at room temperature (25°C) for about 30 min. The enzyme activity was stopped by adding 50 μL of 3M NaOH. ELISA values measured by ELISA reader and expressed as absorbency at 405 nm and absorbency values at least double that of the healthy control (normal serum) were considered positive.
Antiserum titer: The titer of P. carotovorum subsp. carotovorum PCCS63 antiserum was determined by using indirect ELISA. Bacterial suspension was prepared in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) at a concentration of ca. 108 CFU mL–1. Wells were coated by adding 100 μL of the bacterial suspension to the well (2 wells per each antiserum dilution). Serial dilutions of double fold up to 1:1.28×104 of antiserum in serum buffer were used. The absorbance values were measured at 405 nm.
Determination of antiserum efficiency: Indirect ELISA previously described was used to demonstrate the sensitivity of polyclonal antisera to differentiate among all soft rot bacterial isolates; two isolates of P. carotovorum subsp. carotovorum and four isolates from Enterobacteriaceae related genera as follows; P. carotovorum subsp. carotovorum PCCS63 (used for production antisera), P. carotovorum subsp. carotovorum (IPO1949), P. carotovorum subsp. atrosepticum (1007), P. carotovorum subsp. wasabiae (ipp041), D. dianthicola (IPO 2114) and D. chrysanthemi (DSM4610) through their reaction with the produced antiserum against PCCS63 isolate (kindly provided by Prof Dr. Van der Wolf, Plant Research International, Wageningen, Netherlands).
RESULTS
Antisera production confirmation: Results of antiserum production indicated in Table 1 showed that, killed or live bacterial cells of isolate PCCS63 were only positive and interact with homologous antiserum which appeared in ELISA readings. On the other hand, antisera of Pcc LPS and Pca LPS failed to show any relation or positive reaction with PCCS63 cells in ELISA test as compared with normal serum (control).
Optimum antiserum collecting period: Results of indirect ELISA to determine the optimum period to collect the antiserum showed that the first collecting date 7 days after the lasted injection was the best followed by the second collecting date 14 days in both killed and live bacterial antisera Table 2.
Determination of antiserum titer: The best positive ELISA values were obtained with As1 (7 days) up to dilution of 1:1.28×104 then As1 (14 days) 1:6.4×103 and As2 (7 or 14 days) 1:8×102 Table 2.
Efficiency of the antiserum: Indirect ELISA was used for comparing among six isolates causing soft rot and from Pectobacterium related genera: P. carotovorum subsp. carotovorum PCCS63 (used for production antisera), P. carotovorum subsp. carotovorum IPO1949, P. carotovorum subsp. atrosepticum 1007, P. carotovorum subsp. wasabiae ipp041, D. dianthicola IPO2114 and D. chrysanthemi DSM4610. Data presented in Table 3 revealed that our polyclonal antiserum could differentiate among the soft rot bacterial isolates and exposed that a relation among these isolates, in the same way, our polyclonal antiserum had a high sensitivity to react with PCCS63and IPO1949 isolates, less with DSM4610 isolate and finely the least coupling with ipp041 and IPO2114 isolates.
| Table 1: | Indirect ELISA absorbance values (405 nm) of Pectobacterium carotovorum subsp. carotovorum (Pcc) cells and lipopolysaccharides of P. carotovorum subsp. carotovorum and P. carotovorum subsp. atrosepticum (Pca) in its antiserum |
ELISA absorbance values at 405 nm are average of two replicates each, absorbency values of at least double that of the healthy control were considered positive | |
| Table 2: | Indirect ELISA absorbance values (405 nm) of Pectobacterium carotovorum subsp. carotovorum PCCS63 in various dilutions of its antiserum with different collection periods |
ELISA absorbance values at 405 nm are average of two replicates each, absorbency values of at least double that of the healthy control were considered positive, As 1: Antiserum produced from Pcc live cells, As 2: Antiserum produced from Pcc killed cells | |
| Table 3: | Indirect ELISA absorbance values (405 nm) of Pectobacterium carotovorum subsp. carotovorum and related Enterobacteriaceae in various dilutions of its antiserum |
ELISA absorbance values at 405 nm are average of two replicates each, absorbency values of at least double that of the healthy control were considered positive, Pcc 1: P. carotovorum subsp. carotovorum PCCS63, Pcc 2: P. carotovorum subsp. carotovorum IPO1949, Pca 3: P. carotovorum subsp. atrosepticum 1007, Pcw 4: P. carotovorum subsp. wasabiae ipp041 (HQ424871), Ddin 5: Dickeya dianthicola IPO 2114, Dch 6: Dickeya chrysanthemi DSM4610 | |
DISCUSSION
Serological tests have been used to screen seed potatoes for latent populations of Pectobacterium spp. and Dickeya spp., but have generally been found to lack the required specificity and sensitivity. However, results of indirect ELISA to determine the optimum period to collect the antiserum showed that the first collecting date (7 days after the last injection) was the best followed by the second (14 days) so, it could be concluded that the efficiency of antiserum decreased as the collection period advanced. This is in contrast with Mohamed et al. (2008) and contract with those of Hamwieh et al. (1999) and Kayali et al. (2004) thus, could be attributed to genera, species and types of the pathogenic bacteria involved in the tests.
ELISA positive antiserum titer values were obtained up to dilutions of 1:1.28×104. These results were in agreement with those of Alarcon et al. (1995), Hamwieh et al. (1999), Kayali et al. (2004) and Mohamed et al. (2008) who mentioned that, it is important to determine the antiserum titer to reduce its consumption.
According to our results about the efficiency of collected antiserum to detect and differentiate among all tested soft rot isolates, there was a weak reaction between the antiserum and the both strains P. wasabiae and D. dianthicola, this was in the same way of the findings of van Der Wolf et al. (1993), who found false positive results and limitations regarding the sensitivity of detection also remain a problem for serological detection of Dickeya spp. Such antibodies recognize only 68% of isolates (Samson et al., 1990). While, a specify of a monoclonal antibody (6A6) to a fimbrial antigen detected all D. dianthicola isolates tested and some other Dickeya spp. than the polyclonal antibody in a Triple Antibody Sandwich (TAS) ELISA (Singh et al., 2000) as well as, (Vernon-Shirley and Burns, 1992) produced three monoclonal antibodies, which reacted specifically with P. carotovorum and react successfully with 34 serogroup of Pectobacterium atrosepticum and in different results (De Boer et al., 1979, 1987) P. carotovorum subsp. carotovorum isolates are serologically more diverse than P. carotovorum subsp. atrosepticum isolates in contrary with Jones et al. (1993) who couldn’t differentiate between them using polyclonal antibodies. Serological techniques, including the use of monoclonal or polyclonal antibodies can be applied in the case of massive contaminations, but latent infections with low concentration of the pathogen (below 105CFU mL–1) require a more sensitive and reliable method to detect the bacterium in a field sample.
In general, the serological assays do not require expensive laboratory equipment, therefore they can be used everywhere, where simple, cheap and relatively sensitive and specific detection on a large scale is desirable, especially under field conditions.