Bulletin OEPP/EPPO Bulletin 28, 177-1 82 (1998)

Use of PCR as a tool for diagnosis of tobacco rattle tobravirus in seed potatoes

by J. MARTIN

Station de quarantaine pomme de terre, La Motte au Vicomte, 35650 Le Rheu (France)

Diagnosis of tobacco rattle tobravirus (TRV) in potato is difficult because of the existence of non- encapsidated NM-type strains, because similar symptoms are caused by potato mop-top furovirus and physiological internal mst spot and because serological tests are inefficient. The aim of this study was first to compare ELISA and PCR tests. The former was found to be ineffective, so only the PCR technique was in a survey of numerous seed-potato lots (200-tuber samples) from different EU countries. A large disparity was found according to geographical origin, with infection levels of German, Dutch and French seed potatoes of 10.3, 8.5 and 0.6%, respectively. There is a real risk that planting tubers with such infection levels will newly contaminate soil. Besides, TRV is not considered in the EU texts on marketing of seed potatoes, so there is no legal recourse if seed-potato lots are found to be TRV-infected.

Introduction

Tobacco rattle tobravim (TRV) is an important virus transmitted by nematodes of the genera Tnchodorus and Parutrichodorus. It causes economic losses in many crops such as potato, ornamental bulbous crops (tulip, hyacinth, narcissus, crocus, gladiolus and eremurus), lettuce, celery, sweet pepper, sugarbeet (Ploeg, 1992), artichoke and Srachys sieboldi (Japanese artichoke). The symptoms of TRV in the flesh of potato tubers include corky arcs, circles or spots (spraing). These are sometimes prolonged on the epidermis as circular crevasses. Primary symptoms of stem mottle occur on foliage.

Diagnosis of this disease is difficult because a non-encapsidated NM-type strain is common in potato (Harrison & Robinson, 1978) and cannot be detected by serological means. Moreover, the M-type isolates, which are encapsidated, show very large antigenic variability (Harrison etal., 1983). TRV is likewise heterogeneously distributed in potato tubers (Crosslin & Thomas, 1995) and passes with difficulty to daughter tubers. Similar symptoms can be caused by potato mop-top furovirus and by physiological disorder (internal brown spot or internal rust spot of potato tubers), as seen in cultivars such as Marine or Delice.

Robinson (1992) reported on the specificity of the PCR test with primers based on the consensus sequence of TRV RNA 1 (Figs 1 and 2). Weideman (1995) demonstrated the use of this test directly on tubers. The target of this study was to use ELISA and ETR tests on different samples of potato tubers to estimate TRV incidence.

Materials and methods

The work was carried out during a survey of samples from the 1996 potato harvest to detect Meloidogyne chitwoodi or M. fallax. The technique was to cut tubers into 5-10 mm slices to check visually for presence of the nematodes. Each sample consisted of 200 seed-potato tubers. Samples originated in France (159), Netherlands (164) and Germany (58). All tubers showing internal necrosis were analysed to detect TRV.

ELISA test

In order to check the efficacy of the ELISA test, two antisera sold by two firms (antiserum 1 and

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178 J. Martin

5’+ 134 K 7- 29K 16K 3’ RNA1

5’ -/ CP K 29 K * 3’ RNA 2

Figure 1. Genomic organization of tobacco rattle tobravirus. The TRV genome is composed of two RNAs (RNA 1 and RNA 2) encapsidated separately. RNA 1 replicates independently and infects the plant systemically. RNA 2 carries the serological determinants of TRV, is very variable according to strains, and determines the interaction specificity between TRV and nematode vectors (Macfarlane, 1996). In RNA I , proteins 134 K and 194 K could have the function of RNA replicases. Protein 29 K could be a transport protein but could have also a role in symptom induction (Boccara eral., 1986 Hamilton etal., 1987; Ziegler-Graff etal., 1991). Protein 16K could have a function in the infection cycle. RNA 2 carries the gene of the capsid protein (CP). Some strains possess 1 or 2 additional genes coding for proteins 29K and 16 K of RNA 1 (Robinson etal., 1987).

antiserum 2) were tested against 7 TRV isolates: TRVl, TRV2, TRV3 and TRV SOT (isolated from potato), TRV CYS (isolated from artichoke), TRV C (isolated from Japanese artichoke) and TRV PRN (supplied by the Scottish Crop Research Institute).

Nucleic acid extraction

Each tuber slice showing TRV symptoms was crushed with a rollcrusher. Tuber sap was mixed with 250pL of 3 M ammonium acetate and 300 pL of solution containing 2 mL phenol and 1 mL TE.3D (Tris-EDTA, non-idet P-40, lithium dodecyl sulfate and sodium deoxycholate). The mixture was incubated at 65°C for 5 min, then 400 pL of chloroform/isoamyl alcohol (24/1) was added. After

Fig. 2. Electrophoresis gel showing PCR amplification of DNA from different seed-potato samples. MT = scale. Lanes 1-4 Dutch cv. Elkana. Lanes 5-8: Dutch cv. Prevalent. Lanes 9-12: French cv. Spunta. Lane 13: TRV SOT. Lane 14: healthy tuber. Arrows show the size of the amplified DNA.

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TRV in seed potatoes 179

10 min centrifugation at 12,000 g, 300 pL of the aqueous phase was mixed with 1.2 mL 3.6 M LiCl to precipitate RNA (4°C for 1 h). RNA was collected by centrifugation at 12,OOOg at 4°C. After rinsing with 70% ethanol, each pellet was dissolved in 1OpL sterile distilled water.

Reverse transcription

cDNA synthesis was effected with AMV-Reverse Transcriptase (AMV-RT). Primer annealing was carried out in 2 1 pL of a 0.23-pM solution of primer A (Robinson, 1992) and 2 pL of nucleic acids. The annealing mixture was incubated for 3 min at 70°C and chilled. Then 3 pL of reverse transcription mix (3 units AMV-RT (Eurogentec), 2.5 pL AMV-RT buffer and 0.25 pL dNPs (0.05 mM each)) were added to the annealing mixture. The reaction mix was incubated for 1 h at 42°C.

Polymerase Chain Reaction (PCR)

2.5 pL of cDNA were mixed with 24 pL PCR mix (0.2 p M primers A and B (Robinson, 1992). 1.5 mM MgC12, 0.05 m~ each dNTPs, 0,35 unit Taq Goldstar (Eurogentec) and 2.5 pL Taq buffer). The mixture was heated to 94°C for 5min to denature the DNA completely and incubated in a thermocycler (Perkin Elmer 2400) using 22 cycles of 94°C for 30 s, 58°C for 3 min and 72°C for 1 min, followed by 5 min extension at 72°C. The amplification products were stored at 4°C until use. They were detected by electrophoresis in 1% agarose gel. Bands were visualized by ethidium- bromide staining. The expected fragment was 463 nt long but its size was found to change slightly with TRV strains.

Results

ELISA test

The results obtained with the two antisera are shown in Table 1. Antiserum 1 detected only the infected sample supplied by its seller. Antiserum 2 did not detect the infected sample supplied by its seller but did detect the PRN strain of TRV.

PCR test

PCR was first tested with the same strains as ELISA (Table 1). In contrast to the ELISA test, PCR detected all strains.

Table 1. Comparison of ELISA and PCR tests with different TRV isolates

Isolates ELISA test with ELISA test with

antiserum 1 antiserum 2 PCR test

TRV 1 TRV 2 TRV 3 TRV cys TRV SOT TRV c TRV PRN Positive control supplied with antiserum 1 Positive control supplied with antiserum 2

+ -

- + - +

+ -

- =negative result; + =positive result.

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180 J. Martin

All tubers showing TRV symptoms were subjected to PCR. A sample of 200 tubers was said to be contaminated as soon as a TRV-infected tuber was found. Figure 2 shows, as an example, a PCR gel on which 3 out of 4 nucleic-acid extracts from a Dutch seed-potato sample of cv. Elkana gave a positive result for TRV. The same applied to 3 out of 4 extracts from a French sample of cv. Spunta. However, samples from another suspect Dutch cultivar (Prevalent) gave uniformly negative results. TRV was detected by PCR in a sample with typical symptoms but also in one with atypical symptoms, which would be difficult to attribute to a particular virus.

To detect TRV with certainty, 4-5 different nucleic-acid extracts were needed on each tuber. It appeared that the intensity of internal necrosis was negatively correlated with the number of PCR- positive extracts: the more intensive the internal necrosis, the more difficult it was to detect TRV by PCR.

Contamination level of seed-potato lots

Complete results of the tuber cutting inspections, confirmed by PCR, conducted in 1996/1997 on seed-potato lots of Dutch, German and French origin are shown in Table 2. The TRV infestation levels for Dutch, German and French seeds potatoes were 8.5, 10.3 and 0.6%, respectively. The cultivars concerned are also indicated in Table 2.

Discussion

The results obtained show again the inefficiency of the ELISA test for detection of TRV. The reasons for this may be: the presence of the unencapsidated NM strain, great serological variability due to existence of specific combinations between Trichodorus and Paratrichodorus spp. and TRV strains (Brown & Ploeg, 1990). In contrast, the PCR test was efficient and allowed contamination to de detected directly in tubers. This is particularly useful in view of the fact that TRV contaminates daughter tubers with difficulty. As a result, TRV cannot readily be detected in routine post-harvest tuber tests.

The level of contamination in Dutch and German seed potatoes was quite high. For each contaminated lot, at least two contaminated tubers could be observed per 200-tuber sample. This represents a minimum of 1 % contamination, assuming homogeneous distribution. If the plants involved showed visible symptoms in the growing season, or if the tubers gave plants with the same order of visible symptoms in the following season (l%), these lots could not be classified in classes CEEl, CEE2, CEE3 or even A according to the EU seed-potato certification scheme. What is more, planting such lots could lead to long-term contamination of the plots involved. These worrying

Table 2. Summary of TRV-infected seed-potato samples according to origin and cultivar

Origin of samples Germany Netherlands France

Total number of samples (200 tubers) 58 164 159

Number of TRV-contaminated samples 6 (symptoms and PCR)

14 1

Percentage of contaminated samples 10.3% 8.5% 0.6%

Number of contaminated samples per Calla ( I ) , Indira (3). Adora (l), Bea (1). Spunta (1) cultivar Sommergold (2) Elkana (2). Elles ( I ) ,

Florijn ( I ) , Liseta (l), Prevalent (6 ) . Producent (1)

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TRV in seed potatoes 181

figures are not surprising, since Brown & Ploeg (1990) assess that 20% of the Belgian seed- potato production area is contaminated by a TRV-Trichodorus association and 12% of the Scottish area.

Different reasons can be proposed to explain how such a contamination level has been reached in seed potatoes: - if symptoms cannot be seen on the tuber surface (which is generally the case), the only means of

detection is to cut tubers and examine the cut surface visually. This method is rarely used, because it is laborious. As a result, virological tests on pre-sprouted tubers mostly fail to detect TRV;

- the lack of a reliable and routinely applicable detection method has certainly contributed to the fact that TRV problems are little considered;

- other hosts, such as flower bulbs which are widely grown in The Netherlands, support a high multiplication of TRV;

-the soil and climate of The Netherlands and Germany are widely favourable to development of the nematode vectors;

-a decrease in nematicide use (now considered dangerous for the environment) favours the increase of vector populations.

Planting new plots with contaminated seed-potato lots carries the risk of contamination of the plot, by the following mechanisms: - soil on the tuber surface carries viruliferous nematodes which then spread TRV in the plot. This is

relatively unlikely since nematodes do not resist drying; - if the plot already contains nematodes of the genera Trichodorus and Paratrichodorus, they

become viruliferous by feeding on TRV-infected tubers; - TRV-infected weed seeds are carried with the tubers. TRV can indeed be seed-borne in several

species of weeds (Cooper & Harrison, 1973; Locatelli etal., 1978). The contamination of seed potatoes with TRV is of even greater concern because the virus is

not considered in the EU texts on marketing of seed potatoes. There is therefore no legal recourse if seed-potato lots are found to be TRV-infected.

Acknowledgements

We should like to thank H. Marzin and colleagues at LNPV Ntmatologie, Le Rheu (FR) for their valuable contribution to this study.

Utilisation de I‘outil PCR pour le diagnostic du tobacco rattle tobravirus sur pomrnes de terre de semence

Le diagnostic du tobacco rattle tobravirus (TRV) sur pomme de terre est relativement difficile du fait de I’existence de souches NM non encapsidies, de sympdmes analogues 1 ceux provoquis par le potato mop-top furovirus et 1 ceux de la rouille physiologique, et de l’inefficacitk des tests sirologiques. L’Ctude a consist6 dans un premier temps 1 comparer les mCthodes ELISA et PCR. La premike s’est rtvC1Ce inefficace, aussi seule la seconde a Ctt utilisCe lors d’une enqukte sur de nombreux lots de pommes de terre de semence (Cchantillons de 200 tubercules) de provenance UE. I1 a Ctt ainsi mis en Cvidence une grande disparitt en fonction des origines gtographiques avec une contamination respective des tchantillons allemands, nCerlandais et franpis de 10,3, 8,5 et 0.6%. A ces taux de contamination, les risques de contamination du sol par plantation de tubercules infect& ne sont pas ntgligeables. Par ailleurs, le TRV n’est pas pris en compte par les textes communautaires relatifs 1 la commercialisation du plant de pomme de terre, ce qui n’offre aucun recours juridique 1 un acheteur de lots de pommes de terre de semence infectts par le TRV.

0 1998 OEPPIEPPO, Bulletin OEPP/EPPO Bulletin 28, 177-182

182 J. Martin

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0 1998 OEPPEPPO, Bulletin OEPP/EPPO Bulletin 28, 177-182