Pcr detection of blackleg and soft rot causing erwinia in seed potatoes

DNA based detection of blackleg and soft rot disease causing Erwinia
strains in seed potatoes.


Yeshitila Degefu, Sanna Jokela, Erkki-Joki Tokola and Elina Virtanen
The Seed Potato Laboratory, MTT, Agrifood Research Finland, Regional Unit, North Ostrobothnia
Research Station, Tutkimusasemantie 15, FIN-92400 Ruukki, Finland. [email protected]
ABSTRACT

Erwinia carotovora subsp. atroseptica (Eca), Erwinia carotovora subsp. carotovora (Ecc) and
Erwinia chrysanthemi (Ech) are the different sub species of Erwinia that cause the diseases commonly
known as blackleg, aerial stem rot and soft rot on potato. Blackleg and aerial stem rot affect vines
during the growing season, whereas soft rot affects tubers in the field and during transit and storage.
The three species can cause soft rot under cool and moist conditions. E. carotovora subsp. atroseptica
is the major cause of blackleg, a blackening of the stem base of potato plants, which originates from
the mother tuber (Pérombelon and Kelman, 1987). Erwinia carotovora subsp. carotovora mainly
causes aerial stem rot (aerial blackleg), but under high temperatures it has been reported to cause
blackleg like symptoms. E. chrysanthemi also induces blackleg-like symptoms. Until recently E.
chrysanthemi had been mainly confined to warmer climates of Europe, Australia and the tropics. To
date the species has been known to occur in cool temperate regions including Finland. In Finland E.
chrysanthemi the strain has been reported for the first time in 2004 and it appears to spread fast in
certain parts of country (Degefu, unpublished, Pirhonen, personal communication). Although infested
crop residues and rotting tubers are among the important sources of inoculum, latent infections in seed
tuber provide the major source of infection in potato production (Hannukkala and Segertedt, 2004). At
the seed potato laboratory MTT, Ruukki we are carrying out research and services on PCR (DNA)
based detection of latent infection of blackleg and soft rot Erwinia strains. The enrichment of the
bacteria in semi selective liquid medium prior to PCR ( BIO-PCR) is an important initial step for the
success in PCR detection extremely low number of the target bacteria from tubers. The different
strains appear to differ in their ability to compete with other saprophytes and reach the target detection
limit of bacterial population during the enrichment culture of the potato peel extract. Results of prior
PCR enrichment of the bacteria, detection limits of the different strains and preliminary data, from the
analysis of some seed lots from the high grade area of north Ostrobothnia and Åland regions, on the
occurrence of the strains and the new trends of introduction and spread of E. chrysanthemi in Finland
are presented and discussed. Evaluation of the current status of Erwinia diagnostics and areas of future
research are highlighted
Key words. Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. carotovora, Erwinia
chrysanthemi, Blackleg, soft rot, Polymerase chain reaction, Bio-PCR
INTRODUCTION
An important principle for disease prevention is that when the key sources of inoculum for a give
disease are known, effective measures can be implemented to prevent further spread and subsequent
disease outbreaks. A range of traditional and modern techniques is available for detection of known
bacterial pathogens surviving in seed, plant debris, soil, and water and other vectors. As the majority
of bacterial pathogens are transmitted through contaminated seed or propagative materials, detection
of pathogens in these plant materials becomes of vital importance to ensure safe and sustainable
agriculture. Development in the area is necessary as the threat from introduced diseases is constantly
increasing as a result growth in international travel and trade. In addition to phytosanitory issues crop
Maataloustieteen päivät 2006. www.smts.fi biosecurity and strategic prevention of deliberate release of crop pests and pathogens is national security priority, which also demands a rapid and efficient diagnostic technology (Schaad et al. 2003). Accurate routine disease detection requires high level of specificity, sensitivity and speed (López et al. 2003). Potato is subject to pests, diseases and quality problems that increase the cost of production, place the grower art a considerable risk of economic loss, and add environmental quality degradation by virtue of the chemicals used to address the problems. Experiences from North America and Europe indicate that hundreds of billions of dollars are spent on use of pesticides annually to prevent virus, nematodes and late blight problems on potato. There is also risk of withdrawal of some of the most effective chemicals under country’s Food Quality Protection Act. This sudden bans of key pesticides usually have great impact on production unless immediate alternatives to these pesticides are made available. Resistance and disease management practices that reduce exposure of the crop to diseases and pests and reduce the need for pesticides are arguably the best alternative options. Blackleg and soft rot are a seed borne disease of potato caused by the soft rot Edwina namely E. carotovora subsp. atroseptica, E. carotovora subsp. carotovora and E. Chrysanthemi. The three subspecies are responsible for tuber soft rot and are reported to cause blackleg symptoms of varying degree depending on the (Bång, 1989, Pérombelon, 2000). E. carotovora subsp. atroseptica is the major cause of blackleg, a blackening of the stem base of potato plants, which originates from the mother tuber (Pérombelon and Kelman, 1987). Erwinia carotovora subsp. carotovora mainly causes aerial stem rot (aerial blackleg), but under high temperatures it has been reported to cause blackleg like symptoms. E. chrysanthemi also induces blackleg-like symptoms. While E. carotovora subsp. carotovora is widespread and has a wide host range besides potato, E. carotovora subsp. atroseptica is more specifically associated with potato and is prevalent under cold to temperate climates (Hélias et al., 1998).Until recently E. chrysanthemi had been confined mainly to warmer climates of Europe, Australia and the tropics. To date the species has been detected from seed potato cool temperate regions including the high-grade area of Finland (Degefu, unpublished) and other sources such as river water (Pirhonen, personal communications) The economic importance of blackleg is two fold. First it can reduce yield when the disease incidence exceeds 5-10% and second it causes downgrading or rejection of seed potato in the process of certification (Pérombelon, 2000). According to Pérombelon and Hyman (1988) even latent infections of plants can depress potato yield. In addition extensive soft rotting can develop when tubers with blackleg infection are stored. Although infested crop residues and rotting tubers are among the important sources of inoculum, latent infections in seed tuber provide the major source of infection in potato production (Hannukkala and Segertedt, 2004). Under field condition bacteria from rotting mother tubers are transmitted by soil water to the lenticels of progeny tubers (Pérombelon, 1998). Extensive contamination also known to occur during harvest and grading when Erwinia from rotting tubers spread to fresh wounds on large number of tubers. Since the use of chemicals is impractical and there are no resistant potato cultivars control of blackleg and soft rot depends primarily on production of healthy seed, which is best checked by detecting and quantifying tuber contamination by the blackleg and soft rot causing Edwina Staring healthy and staying healthy demands vigilant monitoring and risk management. Detection and diagnostic techniques available for bacteria were based on microscopy, isolation, biochemical characterization, serological techniques, bioassay and pathogenicity tests (Lopez et al, 2003). These are obviously not suitable for routine analysis of large number of samples. Among the major developments in the past two and half decades the Enzyme Linked Immunosorbent assay (ELISA) and the Polymerase Chain Reaction (PCR) is the most significant advance. Currently the detection of phytopathogens has become a very dynamic and evolving world characterised by advances molecular techniques, sequencing, nanotechnology and computer sciences. Automation and electronic data management have become vital to increase the efficiency of for routine analysis of for detection of plant pathogenic bacteria. Effort to develop diagnostic protocols to detect all the 25 potato Maataloustieteen päivät 2006. www.smts.fi pests and diseases listed under EU plant health legislation in a single test is already underway
(tendencies of using more integrated approaches (Alvarez, 2004) including the use of biosensors or an
electronic nose ( Alvarez, 2004, Deisingh, 2004).
Polymerase chain reaction is one of the potential tools for detection plant pathogens. Although further
advances have been made in new variants of PCR such as simple multiplexed nested PCR in a single
closed tube, cooperative PCR and real- time monitoring of amplicons or quantitative PCR ( Lopez et
al 2003), these methods have not been adopted for routine detection soft rot Erwinia in diagnostic
laboratories. PCR has proved to be highly sensitive and more specific than the ELISA method for the
detection of soft rot and blackleg in potatoes (De Boer, 1995) and is expected to serve as an important
tool in the integrated approach for elucidating the spread of blackleg. However, many things need to
be optimised to guarantee its applicability in large-scale routine test.
In this paper we report the use of BIO-PCR whereby viable cells of Erwinia from potato peel extract
are enriched in liquid medium and detected in low levels in seed potato without DNA extraction.
Besides, some preliminary results optimisations of the procedure to enhance the level of detection
sensitivity are presented.
MATERIALS AND METHODS
Bacterial Strains media and culture conditions
E. carotovora subsp. carotovora ( Ecc 107, Ecc 204), E. carotovora subsp. atroseptica (Eca 5b, Eca
1039, Eca 440, Eca 2286) and E. chrysanthemi ( Ech 1A, Ech 04026) test strains kindly provided by
Hannukkala and Joutsjoki, MTT, Jokioinen Crop Protection section were used in the study. In
addition bacterial cultures to test specificity of PCR amplification were also obtained from over one
hundred potato samples obtained from experimental fields and seed potato growers from the North
Ostrobothnia and Åland regions. Bacteria were grown on either CVP (crystal violet polypectate) plates
or NA (nutrient agar). For routine enrichment of the bacteria liquid sodium polypectate medium
(PEM) was used. Bacteria in liquid culture were grown at 27° C shaking at 150 rpm. Bacterial strains
were stored in as glycerol stock at – 80° C.
Sample handling and potato peel extract preparation
Tubers were washed individually in a running tap water to remove soil and other debris and
approximately 3 mm thick peel strips were removed from single tubers with a hand held peeler. The
peel strips ( One round from stolon end to stolon end) were furnished to plant juice press machine
( Meku Erich Pollähne, GmbH, Germany) corrugated roller system. Peel extract running down the
corrugated rolls was collected in a sterile glass test tube. An antioxidant , dithiothreitol (DTT) was
added to a final concentration of 0.075% and the extract was placed on ice for about 10 min to allow
debris and starch to settle before supernatant was pippetted to the polypectate enrichment medium.

Prior PCR enrichment

One hundred microliter of supernatant of the peel extract was transferred to 15 ml Sartedt tubes
containing 5 ml of PEM and incubated at 27 °C for 48 hrs shaking at 150 rpm. The resulting bacterial
cells were collected by centrifugation at 7000 rpm for five minutes and washed twice by resuspending
in 2 ml sterile ultra pure water. The washed bacterial cells were then resuspended in 250 µl of sterile
Maataloustieteen päivät 2006. www.smts.fi ultra pure water and used as PCR template or stored in –20 °C until analysed which is only for a
couple of days.
PCR assay
Primers ECA1f and ECA2r derived from a DNA probe specific to Eca ( De Boer and Ward, 1995) and
Ec001F and Ec001R specific to Ecc and primers Ade1 and Ade 2 specific to Ech were used to
amplify DNA.Dynazyme II DNA polymerase and dNTP mix (Finnzymes, Espoo, Finland ) were
used in a standard PCR reaction assay which employed a one cycle of 2 min denaturation step at 95 C
and 2 min of annealing temperature according to the primer and 2 min of extension at 72 C and 34
cycles of 30 seconds denaturation, 1 min annealing and 2 min extension. Test strains served as
positive controls.
Amplification products were analysed on 1.4% agarose gel and stained with ethidium bromide.
Visualization of the amplification products and documentation of results was carried out using Gel
Doc (BioRad) and quantity one (Bio Rad) software program.
RESULTS AND DISCUSSION
The standard PCR assay when tested with suspension containing 102- 108 cells ml-1 successfully
detected a minimum of about 10 4 cells ml -1of the three strains only with slight differences consistent
to other finding (Hyman et al., 1997). Direct PCR from peel extract with out prior enrichment either
failed or was very inconsistent even using high fidelity Phusion ploymerase suggesting that there is
perhaps extremely low number of the bacterial strain in the tuber or substances of potato origin
inhibitory to the PCR reaction of potato origin are present in the peel extract as it was often speculated
by Hyman and co workers (1997) and other investigators. The BIO-PCR protocol that involves the
prior PCR enrichment of the bacteria by growing the bacteria for 48 hours in a polypectate medium
(PEM) enhanced the sensitivity of the PCR. An added advantage of the enrichment or in general the
BIO-PCR is that although small amplicons from dead bacteria can not be ruled out (Alvarez, 2004),
the major bands result from amplification of the DNA from living cells. The sample is also free from
inhibitory substances as the cells are thoroughly washed before the PCR. In addition no DNA
extraction is needed since the cells will lyse and release the during the initial denaturing step (2 min at
95 °C). Other investigators ( Hyman et al 1997) used CVP plates as medium for enrichment, we have
found that CVP to be inconsistent, less efficient and more expensive as it requires much sodium
polypectate. By using polypectate medium we have managed to reduce the amount of sodium
polypectate by about 12 times ( 1.5 g instead of 18 g per litter of the medium) and obtained an good
and consistent enrichment of the bacteria.
The three strains appear to differ in their ability to compete among themselves as well as with other
saprophytes. Growth of the bacteria (OD660) reduced significantly, compared to the control shake
culture, when potato peel extract was added to a culture containing 103 bacteria of the pure test strains.
Our preliminary result indicated that Eca appear to be less competitive than both Ecc and Ech. The
competitive ability of Eca was improved an anaerobic growth condition apparently because of the
suppression of aerobic saprophytes in the culture.
The optimised PCR protocol is consistent and successfully detects the three soft rot causing Erwinia
strains. We have analysed hundreds of samples. It is evident that seeds could be contaminated by
either one or all the three strains of Erwinia. We have documented new record of E. chrysanthemi
infection in seed potatoes in Finland, also confirmed from report of other labs ( Hannukkala and
Joutsjoki, Pirhonen , personal communication) a strain, which was confined mostly to warmer tropical
regions of the world. This indicates the likely occurrence freeze tolerance strain.
Maataloustieteen päivät 2006. www.smts.fi CONCLUSIONS AND PROSPECTS Pérombelon (2000) emphasized on four important steps in seed-potato health determination or detection of Erwinia which need to be evaluated namely (1) sampling (2) tuber tissue preparation for testing (3) quantification of tuber contamination, and (4) interpretation of the results in terms of blackleg risk assessment. Conclusions drawn from ultrasensistive analytical methods that require only microliter of the sample might often misrepresent the real situation unless sampling that represents realistic order of scale is practiced. The key elements in all sampling procedures are random sampling of the seed lot and blending of primary, composite, and submitted samples (Morrison, 1999). The crucial step in the PCR detection process is the handling of the tubers and preparation of peel extract. Extreme care is needed to avoid cross contamination because of the high sensitivity of the BIO-PCR. One of the main challenges in the diagnosis of Erwinia in seed potato is to separate the target pathogen from numerous saprophytes that overgrow it in culture media The conventional PCR can only indicate the presence or absence of bacteria. There has been significant development in real time PCR technology that has an advantage of determining the absolute quantity of the starting target DNA in a tuber. Our goal in this regard is to develop the quantitative Erwinia diagnostic technology and generate epidemiological data in relation to initial inoculum density of the pathogen to establish threshold infection and tolerance levels for meaningful prediction and certification programs. This rather challenging task. It is hard to accurately from the number of seed-tuber borne bacteria (Bain and Pérombelon, 1990) since growing conditions, which are not known at the time of tuber analysis can have large role in disease development. However, seeds could be designated as high, moderate or low risk with respect to blackleg infection and the estimation of the risk level could be improved by obtaining data under diverse climatic and weather conditions. REFERENCES Alvarez, A.N. 2004. Integrated approaches for detection of plant pathogenic bacteria and diagnosis of bacterial diseases. Ann. Rev. Phytopathol. 42: 339-366. Bång, H. 1989.Prevalence of Erwinia-species in basic seed lots of potatoes grown in Northern Sweden. Acta Agcric. Scand. 39:373-379. De Boer, S.H. & Ward, L.J. 1995. PCR detection of Erwinia carotovora subsp. atroseptica associated with potato tissue. Phytopathol. 85: 854-858. Deisingh, A.K., Stone, D.C. & Thompson, M. 2004. Applications of electronic noses and tongues in food analysis. Int. J. Food. Sci. Tech. 39: 587-604. Hannukkala, A. & Segerstedt M. 2004. Epidemiology, diagnostics and control of blackleg and soft rot on potato Maa- ja elintarviketalous 41. 60 p. MTT, Jokioinen, Finland. Hyman, L.J., Dewasmes, V., Toth, L.K., & Pérombelon, M.C.M. 1997. Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium. Letters in App. Microbiol.25: 143-147 Maataloustieteen päivät 2006. www.smts.fi Hélias, V., Le Roux, A.C., Bertheau, Y., Andrivon, D., Gauthier, J-P., Jouan, B. 1998. Characterization of Erwinia carotovora subspecies and detection of Erwinia carotovora subspecies atroseptica in potato plants, soil and water extracts with PCR-based methods. Euro. J. Pl. Pathol.104: 685-699. López, M.M., Bertolini, E, , Olmos, A., Caruso, P., Goris, M.T., Llop, P., Penyalver, R., Cambra, M. 2003. Innovative tools for detection of plant pathogenic viruses and bacteria. Int. Microbiol.6: 233-243. Morrison, R.H. 1999. Sampling in seed health testing . Phytopathol. 89: 1084-1087. Pérombelon, M.C.M 2000.Blackleg risk potential of seed potatoes determined by quantification of tuber contamination by the causal agent and Erwinia carotovora subsp. atroseptica: a critical review. OEPP/EPPO Bulletin 30: 413-420. Pérombelon, M.C.M & Kelman A 1987. Blackleg and other potato diseases caused by soft rot Erwinias: Proposal for revision of terminology. Plant Diseases 71: 283-285 Pérombelon, M.C.M & Hyman, L.J. 1988. Effect of latent infection by Erwinias on yield. In Scottish Crop Research Annual Report for 1987, pp 102. SCRI, Dundee (GB). Schaad, N.W., Frederick, R.D., Shaw, J., Schneider, W.L., Hicksion, R., Petrtillo, M.D & Luster DG 2003. Advances in molecular based-diagnostics in meeting crop biosecurity and phytosanitory issues. Ann. Rev. Phytopathol. 41: 305-324 Maataloustieteen päivät 2006. www.smts.fi

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