Detection of Dickeya spp. latent infection in potato seed tubers using PCR or ELISA and correlation with disease incidence in commercial field crops under hot-climate conditions
Over a 5-year period (2006–2010), 277 certified, visually healthy potato seed lots, imported from Europe to Israel for commercial use, were tested for Dickeya spp. latent infection by PCR analysis (277 seed lots) and ELISA (154 seed lots). Seeds from these lots were grown in commercial potato fields which were inspected twice a season by Plant Protection and Inspection Services (PPIS). Stem samples were tested for the presence of Dickeya spp. by PCR analysis. PCR and ELISA results from seed lot testing correlated with disease expression in 74 and 83·8% of the cases, respectively. Positive laboratory results with no disease symptoms in the field (‘+lab/−field’ results) comprised 24·7 and 9·7% of the PCR and ELISA analyses, respectively, whereas negative laboratory results with disease symptoms in the field results (‘−lab/+field’) were obtained in 1·3 and 6·5%, of cases respectively. Maximum disease incidence, as well as the number of cultivars expressing disease symptoms, increased over the years of this study, indicating an increase in the prevalence of the disease. Severe disease incidence was observed on cvs Dita, Rodeo, Desiree, Mondial, Tomensa and Jelly. Of the 55 imported seed lots from which disease was recorded in the field, 49 originated from the Netherlands, four from Germany and two from France. None originated from Scotland.
Dickeya spp. (syn. Erwinia chrysanthemi), which are Gram-negative bacteria, are the causal agent of slow wilt and blackleg in potato (Solanum tuberosum). Disease symptoms can vary with climate. The pathogen has been reported to cause pre-emergence tuber decay in semi-arid areas, extensive stem rotting in lowland tropical regions, wilt symptoms associated with only limited stem rot in cool-temperate regions, and wilt but not stem rotting in Israel (Palacoi-Bielsa et al., 2006; Tsror (Lahkim) et al., 2006, 2009).
Until recently, most Dickeya strains found in association with potato blackleg belonged to Dickeya dianthicola (biovars 1 or 7). These strains have a relatively low maximum growth temperature compared with other Dickeya species and seem to be more adapted to European climatic conditions. However, in the last 5 years, Dickeya strains belonging to a new clade different from the six Dickeya species previously described, and which may therefore constitute a new species (‘Dickeya solani’), have been frequently isolated from potato tubers in western Europe and Israel (Parkinson et al., 2009; Sławiak et al., 2009; Tsror (Lahkim) et al., 2006, 2009; Toth et al., 2011).
‘Dickeya solani’ has a higher optimal growth temperature than D. dianthicola; therefore, development and expression of the disease in field crops may be particularly favoured under hot climatic conditions (Toth et al., 2011). Recent outbreaks of Dickeya spp. on potato in Israel, in plants grown from imported seed tubers, are of great concern because of the warm climatic conditions during the spring growing season, which may allow the pathogen, once introduced into the country, to establish and spread (Tsror (Lahkim) et al., 2006, 2009). It may directly affect yield levels and quality, and it could also pose a threat for Israeli agricultural products exported to Europe. Therefore, the pathogen is still considered a quarantine organism in Israel.
Disease symptoms in the recent outbreaks in Israel first appear as wilting of the top leaves, which spreads to the lower leaves and is followed by desiccation. Usually, discolouration of the vascular system in the stem base is observed, followed by external darkening. In severe infections, the stem or whole plant dries out. Symptoms are usually associated with soft rot of the mother tuber, and sometimes (depending on the level of infection), daughter tubers also rot (Tsror (Lahkim) et al., 2006, 2009).
Factors favouring disease development on potato caused by Dickeya spp. are, on the whole, similar to those for Pectobacterium atrosepticum, which also causes blackleg, and include damage and lack of cleanliness at grading, poor soil drainage, presence and increasing level of the pathogen on seed tubers, over-irrigation, wet spring weather, damage at harvest and lack of adequate ventilation at storage (Toth et al., 2011). However, there are factors that may influence disease development differently between Dickeya spp. and P. atrosepticum. These include inoculum level, cultivar susceptibility, speed of migration through the plants’ vascular system, temperature and relative aggressiveness. Unfortunately, there is little published information on many of these factors and their differential roles in disease promotion are largely unknown (Toth et al., 2011). ‘Dickeya solani’ strains were shown to cause more severe losses than D. dianthicola, P. atrosepticum and P. carotovorum subsp. carotovorum (Lojkowska et al., 2010; Toth et al., 2011). However, no difference in average incidences between ‘D. solani’ and D. dianthicola were found in a 3-year field study in the Netherlands (Czajkowski et al., 2010). The aggressiveness of 40 ‘D. solani’ strains evaluated in a tuber maceration bioassay (incubation for 48 h at 30°C) indicated a considerable variability among the tested strains, ranging from 0·4 to 4·0 g of macerated tissue (L. Tsror, unpublished data).
The major source of infection and the most important route of long-distance dispersal of Dickeya spp. as well as other seedborne pathogens are contaminated seed tubers (Tsror (Lahkim) et al., 1999; Toth et al., 2011). Production of pathogen-free seed lots is therefore considered the most effective strategy in controlling pathogen spread. Tuber contamination can occur during plant growth, but harvesting and grading are considered the most susceptible phases. Tuber contamination can be reduced by restricting the number of generations in the field, applying disinfection procedures for mechanical equipment used during harvesting and grading, and disinfecting tubers (Pérombelon, 2002). Control is mainly based on exclusion and reduction of inoculum, as bactericides are generally ineffective. Therefore, the use of seed-testing methods is indispensable. The aim of this work was to study the correlation between PCR and ELISA methods for the detection of latent Dickeya spp. infections and disease incidence in commercial potato crops under hot-climatic conditions.
Materials and methods
Potato lots and field observations
Over a 5-year period (2006–2010), 277 certified potato seed lots (10–100 tons each), imported from Europe to Israel for commercial use, were tested for Dickeya spp. latent infection (Table 1), using a sample of 200 tubers from each lot prior to planting. Potatoes grown from these lots were inspected in the field twice a season by the Israeli Plant Protection and Inspection Services (PPIS). In each inspected commercial field, diseased plants were counted in at least 10 randomly chosen blocks (2 rows wide × 12 m long), which included 500–600 plants. If disease incidence was higher than 10%, an additional 10 blocks were evaluated (for a total of 1000–1200 plants) to get a better estimation of disease occurrence in the field. Samples of diseased plants were tested for the presence of Dickeya spp. using PCR analysis.
Table 1. Country of origin of certified potato seed lots imported to Israel and tested for Dickeya spp.
Seed tuber sampling and enrichment of Dickeya spp.
For each seed lot, four composite subsamples of 50 tubers each were analysed (a total of 200 tubers per lot). Seed tubers were washed in running tap water, surface-sterilized with 0·3% (v/v) hypochlorite for 1 min, and air-dried. After drying, a 0·5-cm-deep tissue sample from the stolon end of each tuber (including both the vascular bundles and the peel) was removed using a sterile scalpel. Fifty stolon ends were pooled and placed in a tube containing 50 mL of polypectate enrichment broth (0·64 g L−1 MgSO4·7H2O, 2·16 g L−1 (NH4)2SO4, 2·16 g L−1 K2HPO4·3H2O, 3·4 g L−1 sodium polypectate, pH 7·2) (Pérombelon & van der Wolf, 2002). The tubes were tightly closed to provide low oxygen conditions and incubated at 28°C for 48 h.
Detection of Dickeya spp. in seed lots
Subsamples (of 50 tubers each) were used to detect Dickeya spp. latent infection through enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR). ELISA was performed in microplates (NuncMaxiSorp™; two wells per sample), using polyclonal antibodies Ech-Co and Ech-AP (Plant Research International, Prime Diagnostics) or Product code 1080 (Neogen Europe) according to the manufacturer’s instructions. The absorbance was measured with an automatic ELISA reader (EL ×800 Absorbance Microplate Reader, Bio Tek) at 405 nm (A405) and, after 1 h, A405 values over twofold the mean value of negative controls (supplied by the commercial ELISA sets) were considered to be positive. Dickeya dianthicola was supplied with the commercial ELISA sets as a positive control. Pectobacterium atrosepticum and P. carotovorum reference strains (obtained from A. Lees, SCRI, Scotland) were also included in these tests because these pathogens may also be present on potato crops.
PCR was conducted according to Nassar et al. (1996), using ADE1/ADE2 primers (ADE1: 5′-GATCAGAAAGCCCGCAGCCAGAT-3′, ADE2: 5′-CTGTGGCCGATCAGGATGGTTTTGTCGTGC-3′). PCR was performed with ReddyMix PCR MasterMix (Thermo Scientific) with initial denaturation at 94°C for 4 min, followed by 40 cycles consisting of incubations for 30 s at 94°C and 1 min at 72°C, with a final extension of 10 min at 72°C. The expected fragment length of the amplicons was 420 bp. Amplified DNA was detected by electrophoresis on a 1·5% agarose gel in 0·5 × TAE (40 mm Tris–acetate, and 1 mm EDTA at pH 8·0) buffer and staining with ethidium bromide. Genomic DNA was extracted from the bacterial pellet using Maxwell 16 DNA kit for Gram-negative bacteria (Promega) or GenElute Bacterial Genomic DNA kit (Sigma), according to the respective manufacturers’ protocols. Approximately 100 ng of DNA was used in the PCR assays which were conducted as described above.
Detection of Dickeya spp. in diseased potato plants
Diseased potato plants showing wilt of the top leaves with or without stem pith necrosis, usually with brown internal necrosis at the stem base, were collected from inspected commercial potato fields. Stems were surface-sterilized in 0·3% (v/v) NaOCl for 3 min, washed in running tap water, and dried in a laminar flow cabinet for 2 h. Ten 20-mm-long segments taken from the stem (starting from the base up to 20–25 cm) of each plant were homogenized with 10 mL sterile distilled water in an Ultraturrax blender (Janke Kunkel) for 60–90 s at 4°C. Suspensions were streaked on modified crystal violet pectate (CVP) selective medium (Hyman et al., 2001) and incubated at 33°C for 48–72 h (Pérombelon & Hyman, 1986). Cells from suspected colonies were collected from the medium using a sterile toothpick and grown in nutrient broth. Genomic DNA extraction and PCR analysis were conducted as described above.
Planting tubers with Dickeya spp. latent infection for experimental field observation
In 2009–2010, a sample of 100 tubers from 20 commercial seed lots was manually planted in experimental observation plots established in two locations (Gilat Research Center and Kibbutz Zeelim) of the Negev region in southern Israel, the largest potato-growing area in Israel. Seed lots for the experimental observation were selected as positive for Dickeya spp. infection based on PCR analysis. These experimental plots were surveyed on a weekly basis, starting from 40 to 60 days after planting. Dickeya spp. infection of wilted plants was confirmed using PCR analysis as described above.
PCR or ELISA analyses were considered positive when one or more subsamples from each lot tested positive for Dickeya spp. Disease expression in the field was classified into three levels: 0, 0–1 and >1% disease incidence. Average disease incidence was calculated for fields with over 1% wilted plants. Disease incidence percentages were transformed to arcsine values and analysed statistically with JMP software (SAS Institute Inc.). Data were subjected to one-way analysis of variance (anova) and separated by Student’s multiple range test (P = 0·05).
Correlation between PCR analysis and disease incidence in commercial potato fields
During the 5-year study (2006–2010), 277 imported commercial potato seed lots were examined for Dickeya spp. latent infection using PCR analysis: 178 of the inspected seed lots (64·3%) were found to be negative for Dickeya spp. and 99 seed lots (35·7%) were found to be positive (Table 2). In 77·6% of the tested seed lots, PCR analysis of seed tubers correlated with disease expression in the field (61% negative in laboratory test with no disease symptoms in the field; 16·6% positive in laboratory test with disease symptoms in the field). However, in 19·1% of the tested seed lots that were PCR-positive, no disease symptoms were observed in the field (‘+lab/−field’), and in 3·25% of the tested seed lots that were PCR-negative, disease symptoms were observed in the field (‘−lab/+field’) (Table 2).
Table 2. Correlation between PCR analysis of certified seed lots and disease expression in commercial field crops
| No. of lots (%)||169d (61·0e)||6 (2·2)||3(1·1)||178 (64·3)||9·4 a|
| No. of lots (%)||53 (19·1)||21 (7·6)||25 (9·0)||99 (35·7)||12·7 a|
|Total||222 (80·1)||27 (9·8)||28 (10·1)||277|| |
Comparison between PCR and ELISA analyses
PCR and ELISA detection methods were compared for 154 of the 277 imported seed lots (Table 3). Seed analysis by PCR and ELISA correlated with disease expression in the field in 74% and 83·8% of the cases, respectively. Positive laboratory results with no disease symptoms in the field (‘+lab/−field’) were 24·7 and 9·7%, for PCR and ELISA analyses, respectively, while negative laboratory results with disease occurrence in the field (‘−lab/+field’) were 1·3 and 6·5%, respectively. Disease incidence (>1%) from lots defined by PCR and ELISA as positive for Dickeya spp. was 10·5 and 13·0%, respectively, while disease incidence (>1%) from lots defined by PCR and ELISA as negative for Dickeya spp. was 0 and 10·0%, respectively (Table 3).
Table 3. Correlation between PCR or ELISA analysis and disease expression in commercial field crops
| No. of lots (%)||88d(57·1e)||2 (1·3)||0 (0·0)||90 (58·4)|| |
| No. of lots (%)||38 (24·7)||12 (7·8)||14 (9·1)||64 (41·6)||10·5|
| No. of lots (%)||111 (72·1)||8 (5·2)||2 (1·3)||121 (78·6)||10·0 a|
| No. of lots (%)||15 (9·7)||6 (3·9)||12 (7·8)||33 (21·4)||13·0 a|
|Total||126 (81·8)||14 (9·1)||14 (9·1)||154|| |
Association between disease incidence and climatic conditions
Disease incidence increased over the years (Table 4). Average disease incidence was highest in 2010 (Table 4), but not significantly different from that in the previous years. Meteorological records showed that the average, maximum and minimum temperatures were higher in 2010 than in 2007–2009 (data not shown), indicating that the climatic conditions may have an important effect on disease expression. In addition, the number of cultivars expressing disease symptoms in the field increased over the years (from one cultivar reported in 2006 to 11 cultivars in 2010; Table 4), indicating spread of the disease in Europe and Israel.
Table 4. Average and range of disease incidence and cultivars expressing disease symptoms in commercial field crops
|2006||12||5||4||8·0 a (1–13)||Mondial|
|2007||18||1||5||12·0 a (1–20)||Mondial, Desiree|
|2008||70||10||2||10·0 a (1–20)||Mondial, Bellini, Virgo|
|2009||58||8||8||9·25 a (1–30)||Mondial, Nicola, Dora, Sante, Santana|
|2010||64||3||9||17·7 a (1–36)||Alpha, Chopin, Dita, Dora, Jelly, Laperla, Laura, Rodeo, Vivaldi, Rossana, Tomensa|
|Total||222||27||28|| || |
Association between disease incidence and origin of seed tubers
Of the certified seed lots imported to Israel, 176 originated from the Netherlands, 45 from Scotland, 36 from France and 20 from Germany (Table 1). Interestingly, disease incidence in commercial fields was significantly higher for seed lots originating from Germany (P < 0·05; Tables 5 & 6). Out of 55 disease-expressing lots, 49 originated from the Netherlands, four from Germany and two from France, while none originated from Scotland.
Table 5. Disease expression in commercial field crops or experimental plots from seed lots of different country of origin
|The Netherlands||127||24||25||10·8 b|
Table 6. Average disease incidence of different potato cultivars planted in commercial fields
|Alpha||3||1||0||4||–||N (3); G (1)|
|Nicola||15||3||4||22||4·5 b||N (19); S (3)|
|Mondial||27||12||7||46||11·7 ab||N (44); F (2)|
|Desiree||23||0||4||27||12·5 ab||N (13); S (14)|
|Total||201||26||22||249|| || |
Association between disease incidence and cultivar
PCR laboratory results were negative (data not shown) and no disease symptoms were observed in the following cultivars: Almera, Charlotte, Alliance, Anabelle, Lady Crystal, Valor and Winston (Table 6). Very low disease incidence (<1%) was observed in cvs Alpha, Mozart and Rossana. Low to medium disease incidence (2–10%) was observed in cvs Bellini, Vivaldi, Nicola and Santana. High disease incidence (>10%) was observed in cvs Dita, Rodeo, Desiree, Mondial, Tomensa and Jelly (Table 6). Cultivars represented by three lots or less were not included in the statistical analysis. Nevertheless, it should be noted that in a single lot of cv. Laura originating from Germany (during 2010), disease incidence reached 50% (data not shown).
Tubers with Dickeya spp. latent infection planted for observation
A sample of 100 potato seed tubers with Dickeya spp. latent infections (as detected by PCR) from 20 commercial seed lots was manually planted in experimental observation plots. Disease symptoms were observed in 17 seed lots, 15 of which expressed an average disease incidence of >1% (Table 7). Disease symptoms were not observed in three of the lots.
Table 7. Disease expression in experimental plots 2009–2010
|No. of lots (%)||3c (15d)||2 (10)||15 (75)||8·7||Laperla, Laura, Labella, Vivaldi, Jelly, Dita, Rodeo, Dora, Santana, Nicola, Mondiale|
Data demonstrated that disease incidence associated with Dickeya spp. in Israel has increased over the last 5 years, probably due to the favourable prevailing climatic conditions, causing economic damage (yield reduction of 20–25% at disease incidence >15%; Tsror (Lahkim) et al., 2009). Expression and damage occur primarily in the spring season, when only imported seed tubers are used (25 000 tons annually). The number of imported seeds with latent Dickeya spp. infections is on the rise because of the intensive spread of Dickeya spp. in Europe (Tsror (Lahkim) et al. (2006, 2009, 2010); Toth et al., 2011). Considerable effort is therefore being invested in developing a method for the detection of latent infections in imported seed tubers. Production of pathogen-free seed lots is considered the most effective strategy in controlling the spread of this pathogen (Czajkowski et al., 2009). Pre-PCR processing in the method implemented in the current work included: surface sterilization, sampling of the stolon ends from 200 tubers per lot and incubation in enrichment broth. In agreement with the sampling here (0·5-cm-deep tissue from the stolon end including both the vascular bundles and the peel), Czajkowski et al. (2009) demonstrated that a high number of bacteria are located in the peel and stolon end, and in tuber samples up to 0·5 cm away from the stolon end. Moreover, when coupled to an enrichment step, PCR and ELISA have been proven to be sensitive for Dickeya spp. detection (Degefu et al., 2009).
The protocol developed to detect latent infection with Dickeya spp. in potato seed lots was used on a large scale, providing a first demonstration of its correlation with disease expression in the field. PCR analysis was shown to correlate with disease expression in approximately 78% and 85% of cases in commercial fields and experimental plots, respectively (Tables 2 & 7). However, in both commercial fields and experimental plots, approximately 15–20%‘+lab/−field’ results were obtained (Tables 2, 3 & 7). Disease expression in potato plants depends on various factors, such as cultivar, initial inoculum present in the seeds, soil moisture, soil type, pH and temperature (Pérombelon, 2002; Toth et al., 2011). Therefore, it is possible that detectable amounts of the pathogen at low levels may not always express disease symptoms. Furthermore, 3·3%‘−lab/+field’ results were obtained in commercial fields. A sample size larger than 200 tubers might reduce the proportion of ‘−lab/+field’ results (especially in large lots of over 25 tons) and allow detection of lower levels of infection.
PCR and ELISA detection methods were compared for 154 of the 277 seed lots. The number of ‘+lab/−field’ results was higher in the PCR analysis (24·7%) than with the ELISA (9·7%) (Table 3), probably due to the higher sensitivity of the PCR method (Diallo et al., 2009). However, ‘−lab/+field’ results were higher for ELISA (6·5%) than for PCR (1·3%), indicating that the ELISA detection method presents a greater risk of pathogen dissemination via the planting of infected potato seed tubers.
Of the 55 infected seed lots, 49 originated from the Netherlands (Table 5), whereas none of the seed lots originating from Scotland were infected, although susceptible cultivars were imported (cvs Nicola and Desiree, Table 6). Disease incidence in commercial fields was significantly higher for seed lots originating from Germany (Tables 5 & 6), probably due to cv. Jelly, which expressed the highest disease incidence of all of the cultivars.
Crop losses can occur and the reputations of seed growers, suppliers and exporters can be damaged if seeds exported to warmer climates break down due to the presence of ‘D. solani’. With the emergence and spread of ‘D. solani’, and the effects of climate change, more frequent losses of this kind can be expected (Toth et al., 2011).
Disease incidence and the number of infected cultivars increased over the years of the current study (Table 4), reflecting the increasing prevalence of the disease in Europe, in accordance with other reports (Toth et al., 2011). Furthermore, disease incidence was highest in 2010 (Table 4), which was significantly warmer than any of the previous years of the study period (data not shown), supporting the fact that temperature has a great impact on disease expression. This is similar to the situation in Finland in which increasing temperatures were shown to be a major factor (Laurila et al., 2008, 2010). It should be noted that precipitation in the northern and western Negev regions, where potatoes are grown, is <200 mm annually, and it is therefore assumed that temperature plays a major role in disease occurrence under growth conditions in warm-climate areas. A striking example of this effect occurred with cv. Jelly: potato plants with no disease symptoms developed 30% disease incidence after a short heat wave of over 38°C (data not shown).
‘Dickeya solani’ is considered a quarantine pest in Israel due to the prevailing climatic conditions which favour its growth. The introduction of this pathogen with imported seed tubers is of great concern, due to its potential establishment and spread to other hosts, including weeds. The authors recently reported on latent infection of Cyperus rotundus sampled from a field in which infected potato plants had been observed. Infection was caused by the new Dickeya sp. clade (Tsror (Lahkim) et al., 2010) which constitutes a major threat in potato-growing areas in both Europe and Israel.
The authors thank R. Klienerman and his team from PPIS for their contribution in monitoring disease incidence in the commercial field crops during this study, and Camille Vainstein for editing the manuscript. This research was supported by the Vegetable Council and by the Chief Scientist of The Ministry of Agriculture and Rural Development, Israel. Contribution from the Agricultural Research Organization, Institute of Plant Protection, Bet Dagan, Israel is also acknowledged.