Explanitary notes on seed transmission of Pepino mosaic virus ...

Explanatory <strong>notes</strong> on seed transmission of Pepino mosaic virus (PepMV)The possible seed transmission of PepMV is still a matter of debate despite earlier reports that seedtransmission was possible (Krinkels, 2001; Ling and Carpenter, 2005; Cordoba-Selles et aI., 2007).While seed transmission levels were generally found to be very low, these studies were only performedon limited numbers of seeds. The PEPEIRA project allowed us to perform these studies on a much largerscale thereby increasing the confidence interval of the tests.Starting from a putative seed transmission rate of 1 in 10.000, it was calculated that testing ofapproximately 100.000 seeds in batches of 10 seedlings should result in a confidence interval for theestimate of seed transmission of 2.5 E-6 to 0.0006.Tomatoes were collected from a greenhouse tomato trial infected with a mixture of the Europeantomato strain and the Chili-2 strain of PepMV. Seeds were harvested in three batches at 8, 12 and 15weeks after initial mechanical inoculation of the crop.Following harvest seeds were manually separated from the tomato pulp and collected in containers. Theresulting volume of seeds and leftover pulp was diluted ½ with tap water. Subsequently, to enzymaticallyremove the seed pectin layer, pH was adjusted using citric acid (pH 4, final ratio 1/15) and the enzymepectinase (Pectinex, final concentration 0,25%) was added to the diluted pulp. During a 3 hour period,the mixture was incubated at 28°C and stirred every 30 minutes. Afterwards the seeds were separatedfrom the mixture by sieving and subsequently they were rinsed thoroughly with tap water (figure 1a). Theseeds were centrifuged and washed again by rinsing with tap water. Finally, the seeds were centrifugedand dried during a 24h period in an oven at 26°C, with maximal ventilation. After drying the watercontent of the seeds was measured. Seeds were considered as ready for shipment when the watercontent was below 6%.A total of over 100.000 seeds was harvested and all seeds were shipped and sown within six weeksafter harvest. Seed batches were distributed among ten consortium partners. A total number of 87.780seedlings were obtained which were tested in batches of 10 seedlings in a standard ELISA protocolusing a commercially available antiserum (Prime Diagnostics) against the European tomato strain ofPepMV.Twenty-three pooled batches of plants were found positive in ELISA tests meaning that 23 seeds (oneseed per pooled batch) gave rise to infected seedlings. These test data suggest a minimumtransmission percentage of 0.026% depending on the time interval between the virus infection of themother plant and the harvest of the seeds. Remarkably only 1 infected pooled batch was found in thefirst seed batch (harvested 8 weeks after inoculation of the crop), 3 positive pooled batches were foundin the second seed batch and a total of 19 infected pooled batches in the third seed batch harvested 15weeks after inoculation of the crop. The results show that seed transmission of PepMV can occurs,although at a very low level, and that the risk of seed transmission appears to increase with time afterinfection.Since 2001 (Commission Decision 2001/536/EC), EU regulations are in force directed at preventingintroduction of PepMV into the European Union on tomato seed (prevailing law is the commissiondecision 2004/200/EC). The EU decision now in force demands acid extraction of the seed and eitherproof of absence of PepMV in the area or in the seed crop (inspection during active growth) or in theseeds (lab-test).Following the EU-directive all major seed companies have implemented rigorous seed crop screeningprograms for PepMV. In practice, tomato seeds are harvested from PepMV-free plants, while seedtreatments and seed tests provide additional protection against the (small) risk of seed transmission.In contrast to common practice, the seed transmission test as performed by the PEPEIRA consortiumbasically represents a ‘worst case scenario’. Not only were seeds harvested from an infected crop up toa period of 15 weeks after infection but harvested seeds were only marginally cleaned using pectinase.

Although this treatment is done in an acidic environment it is in no way comparable to the acid treatmentor other disinfection methods used by seed companies as quality assurance measures.The results from the large scale PEPEIRA seed transmission study confirm earlier reports onlow levels of PepMV transmission through seeds harvested from PepMV infected plants. Theoverall low level of transmission (0.026%) was obtained in tests performed under ‘worst casescenario’ conditions which are not comparable with the common practice as used by seedcompanies in compliance with Directive 2004/200/EC.ReferencesKrinkels M . PepMV causes sticky problems…. Prophyta May 2001: 31-33Ling K and Carpenter L (2005). Pepino mosaic virus, an emerging disease in tomato greenhouseproduction worldwide; is seed responsible? Proc 1sr IC on tomato diseases. Eds. MT Momol, Ji, P. andJones JB. Acta Horticultura 695. ISHS 2005Cordoba-Seles C., ….(2007) Seed transmission of Pepino mosaic virus and efficacy of tomato seeddisinfection treatments. Plant Disease 91: 1250-1254.