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Potato Research 45 (2002) 37-43

Alternative hosts for potato mop-top virus, genus Pomovirus and its vector Spongospora subterranea f.sp. subterranea B I R G I T T E A.B. ANDERSEN, MOGENS N I C O L A I S E N and STEEN LYKKE NIELSEN Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, DK-4200 Slagelse Accepted for publication: 3 May 2002 Additional keywords: hydroponic system, weeds, Denmark

Summary Seventeen weed species common in the potato fields in Denmark were grown in a hydroponic system infested with viruliferous zoospores of Spongospora subterranea f.sp. subterranea carrying potato mop-top virus (PMTV). The plants were examined for infection with PMTV and S.s.s. using DAS-ELISA and based on visible symptoms. Only two weed species were found to be infected with PMTV, Chenopodium album and Solanum nigrum, whereas 13 became infected with &s.s.C. album was infected with PMTV by mechanical inoculation were the infection became systemically and the leaves showed necrotic spots. S. nigrum became locat infected with PMTV by mechanical inoculation. By inoculation with viruliferous S.s.s. the roots of S. nigrum became infected but in three weeks PMTV had not spread into the top of the plants. Nicotiana benthamiana was used as a control susceptible to S.s.s. and PMTV.

Introduction Potato mop-top virus (PMTV) provokes spraing in potato tubers by primary infection. Its vector Spongospora subterranea (Wallr.) Lagerh. f.sp. subterranea (S.s.s.) causes powdery scab on potato tubers and root galls. PMTV was first described in Scotland and Northern Ireland in 1965 (Calvert & Harrison, 1966). In Denmark, PMTV was first recorded in 1986 (Anon., 1986). A survey for PMTV carried out in 1997 (Nielsen & Molgaard, 1997) revealed that PMTV is widespread in all the important potato growing areas in Denmark and that S.s.s. also is widespread. Spraing provoked by PMTV is a serious and increasing problem in Danish potato growing while powdery scab seldomly appears as a problem. The host range of PMTV presently known includes plants in the following three families: Solanaceae, Chenopodiaceae and Aizoaceae (Jones & Harrison, 1969; Harrison & Jones, 1970; Jones & Harrison, 1972). The host range of S.s.s. is broader and hosts are found in Aizoaceae, Apiaceae, Asteraceae, Boraginaceae, Brassicaceae, Caryophyllaceae, Chenopodiaceae, Coniferae, Fabaceae, Lamiaceae, Papaveraceae, Plantaginaceae, Poaceae, Polygonaceae, Ranunculaceae, Resedaceae, Solanaceae, Urticaceae (Wiirzer, 1964 cf. Karling, 1968; Jones & Harrison, 1969; Jones & Harrison, 1972; Foxe, 1980; M~ik~irfiinen et al., 1994). Only a few of the known host plants are common in Danish potato fields. It is assumed that PMTV survives in the field inside resting spores of S.s.s. over the years when potatoes are Potato Research 45 (2002)

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BIRGITTE A.B. ANDERSEN, MOGENS NICOLAISEN AND STEEN LYKKE NIELSEN

not grown (Calvert, 1968). Viable resting spores containing PMTV have been found in a field 18 years after the last potato crop (Calvert, 1968). Another strategy of survival might be in perennial weeds hosting both PMTV and S.s.s. The spread of PMTV in Denmark has been rapid. Symptoms of PMTV in seed tubers imported from Denmark to Israel in 1967 have been reported (ZimmermannGries, 1972), but it was first in 1986 that PMTV was officially reported in Denmark (Anon., 1986). Eleven years later a survey revealed that the virus was widespread (Nielsen & Molgaard, 1997). Weed hosts may have been involved in this fast propagation of PMTV. To test this hypothesis a search for alternative hosts of PMTV and S.s.s. among weeds common in Danish potato-growing areas was carried out. A hydroponic system (Arif et al., 1995) was established to obtain root infections with viruliferous zoospores of S.s.s. as under field conditions. Furthermore, the susceptibility of the host plants to mechanical inoculation with PMTV was tested. As Nicotiana benthamiana was used as virus infector plant, investigations of the timing of appearance of PMTV in the roots of this host plant after leaf inoculation were included. The hydroponic system was chosen because a high infection pressure was easy to maintain, experiments could be replicated under nearly identical conditions and it was easy to take root samples compared to plants grown in soil. Previous investigations of the host range of PMTV and S.s.s. have been carried out in infected fields, in pot trials using infected soil or by adding viruliferous resting spores scraped off infected tubers (Wtirzer, 1964 cf. Karling, 1968; Jones & Harrison, 1969; Harrison & Jones, 1970; Jones & Harrison, 1972; Foxe, 1980; Mfik/ir/iinen et al., 1994). In these experiments the number of resting spores that actually germinated was unknown. Materials and methods

Isolates o f P M T V and S. subterranea Infection with a virus-free Danish isolate (from Tylstrup Research Station) o f S. subterranea was established in roots of N. benthamiana by growing the plants in water with sporeballs of S.s.s. added (Merz, 1989). A Danish isolate of PMTV (54-1) baited from a soil sample, using N. benthamiana as bait plant, was used. To maintain the isolate, leaves of the bait plant were macerated in A+G-buffer (40 g/L PEG 6000, 4.8 g/L Na2HPO4, 2HzO, 0.4 g/L KH2PO4, pH 7.7) and inoculated mechanically to carborundum dusted leaves of new N. benthamiana plants. DAS-ELISA DAS-ELISA was performed by a standard procedure (Clark & Adams, 1977) using polyclonal antiserum produced against PMTV at The Danish Institute o f Agricultural Sciences (DIAS). A sample was considered positive when the absorbance value at 450 nm was greater than 0.10 and greater than twice the mean of the healthy controls (Samson et al., 1993).

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Potato Research 45 (2002)

ALTERNATIVE HOSTS FOR POMOVIRUS AND ITS VECTOR

Weed species Seventeen weed species common in Danish potato fields were included: Artemisia vulgaris L., Cirsium arvense (L.) Scop., Chamomilla suaveolens (Pursh) Rydb., Chenopodium album L., Galium aparine L., Geranium pusitlum L., Lapsana communis L., Matricaria inodora L., Poa annua L., Polygonum avicular L., P. convolvulus L., Rumex acetosa L., R. acetosella L., Solanum nigrum L., Sonchus arvensis L., Urtica urens L. and Viola tricolor L. Nicotiana benthamiana Domin. was used as positive control for presence of infection in the hydroponic system. Seeds were obtained from DIAS and the Botanical Garden of the University of Copenhagen. The hydroponic system A hydroponic system as described by Arif et al. (1995) was established using a flooding and draining period of 6 h intervals. The hydroponic system consisted of a vessel connected to a buffer reservoir and a pump allowing flooding and draining. A plastic top plate with holes for 54 mm pots seals the vessel and gave each vessel a capacity of 20 plants. Using this system, a high humidity was maintained in the vessel during the draining period. Viruliferous S.s.s. was established by placing N. benthamiana systemically infected with PMTV in the hydroponic system together with N. benthamiana infected with S.s.s. The hydroponic system was placed in a growth chamber at 20 ~ with a photoperiod of 16 h. Afterwards four inoculum plants of either N. benthamiana or N. debneyi infected with viruliferous S.s.s. were used per vessel. Fresh nutrient solution (Merz, 1989) was added to the vessel for each experiment but the vessel was not sterilised between each experiment. Experimental setup for distribution o f P M T V in N. Benthamiana Eighteen plants of N. benthamiana were divided into three groups and inoculated with PMTV at the 2 leaf stage using A+G buffer. As control 6 N. benthamiana were inoculated with pure A+G buffer. One week after inoculation and subsequently every week 1 plant from each o f the 4 groups was tested for presence of PMTV by ELISA. From each plant a sample of 0.5 g tissue was taken from a top leaf and a leaf from the middle of the plant and 0.5 g of roots. The plants were grown in a glasshouse at 20-24 ~ and the experiment was replicated once. Experimental set up for alternative hosts for P M T V and S. subterranea The experimental design for each weed species was 3 plants per vessel in 3 vessels, and this was replicated once with a 3 weeks interval. In each vessel 4 weed species (3 plants from each) together with 4 inoculum plants and 4 control plants of N. benthamiana were grown in a randomised layout, for three weeks. After 16 days in the hydroponic system two small pieces, about 2 cm each, of the root of each plant were stained in 0.05% trypanblue in lactoglycerol and examined under a light microscope for the presence of zoosporangia of S.s.s. After 3 weeks all plants were harvested and tested for infection of PMTV by ELISA. From each plant a pooled leaf sample consisting of pieces from top, middle and basic leaves and a root sample were tested. Each sample had a weight between 0.1 and 0.5 g. Potato Research 45 (2002)

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BIRGITTE A,B. ANDERSEN, MOGENS NICOLAISEN AND STEEN LYKKE NIELSEN

Mechanical inoculation o f plants with P M T V Three plants per weed species and as control 3 N. benthamiana were mechanically inoculated with PMTV on 2 leaves per plant at the two-leaf stage. The plants were grown in a glasshouse at 20-24~ for 3 weeks before roots, the inoculated leaves and the top leaves of each inoculated plant were tested for PMTV by ELISA. Results

Distribution o f P M T V in N. benthamiana iV. benthamiana was mechanically inoculated with PMTV and after several time intervals, top and middle leaves and roots were tested for the presence of virus. The results are shown in Table 1. Two weeks after inoculation PMTV was found in all parts of the plants and at the same time it was possible to see symptoms in the non-inoculated leaves. All control plants were negative in the ELISA tests and did not show any symptoms. Alternative hosts f o r P M T V and S. subterranea Of the 17 weed species tested only S. n igrum (4 out of 9 plants tested) became infected with PMTV in the hydroponic system. The infection was restricted to the roots. Two plant species, S. nigrum and C. album, were susceptible to mechanical inoculation with PMTV and developed local infections in leaves. S. nigrum showed no symptoms while C. album developed local necrotic spots. The results are shown in Table 2. Most of the weed species tested were infected by S.s.s.. Zoosporangia were detected in the roots of 13 of 17 species but with large variations in infection rates. In some species like Chamomilla suaveolens and Solanum nigrum alt plants were infected, in contrast in Matricaria inodora only two out of the 18 plants were infected. The 6 species of Compositae tested varied in infection rate from 18/18 for Chamomilla suaveolens to 0/18 for Cirsium arvense. No root infection was detected in Rumex acetosa whereas the closely related Rumex acetosella and the two other species of Polygonaceae showed high infection rates. The only monocotyledon represented, Poa annua, was not infected. Table 1. Inoculation ofN. benthamiana with PMTV. Results shown as the number of samples that were PMTV-positive in ELISA per the total number of tested plants 1\6 weeks after inoculation. The data are combined from two experiments. Weeks after inoculation

1

2 3 4 5 6

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Part of plant Top leaf

Middle leaf

Root

0/6 5/6 5/6 6/6 5/6 6/6

0/6 1/6 5/6 6/6 6/6 6/6

0/6 6/6 5/6 6/6 4/6 6/6


ALTERNATIVEHOSTSFOR POMOVIRUSAND ITS VECTOR Table 2. Infection of 17 weed species with S.s.s and PMTV in a hydroponic system and infection with PMTV by leaf inoculation. Results show the number of plants infected with S. subterranea or PMTV out of the total number of plants exposed to infection. Weed species

Infection with S.s.s. in the hydroponic system

Infection with PMTV in the hydroponic system

Infection with PMTV by sap-inoculation

Artemisia vulgaris Cirsium arvense Chamomilla suaveolens Chenopodium album Galium aparine Geranium pusillum Lapsana communis Matricaria inodora Poa annua Polygonum avicular P. convolvulus Rumex acetosa R. acetosella Solanum nigrum Sonchus arvensis Urtica urens Viola tricolor

1/ 18 0/18 18/18 1/18 7/18 11/18 0/18 2/18 0/18 12/18 17/18 0/15 13/15 18/18 12/15 17/18 3/18

O~18 O~18 0/18 0/18 O~18 0/18 0/18 0/18 0/18 O~18 0/18 O~15 O~15 4/18 O~15 O~18 0/18

O~18 O~18 0/18 9/18 O~18 0/18 0/18 0/18 0/18 O~18 0/18 0/18 O~18 7/18 O~18 O~18 0/18

All control plants of N. benthamiana contained zoosporangia in roots and were PMTV-infected in all plots.

Discussion The investigations of the distribution of PMTV in mechanically inoculated N. benthamiana plants showed that PMTV becomes systemic within 2 weeks post-inoculation and that the virus could also be found in the roots at that time. This result was used in the second experiment to establish the period for propagation of PMTV in N. benthamiana before the infector plants were placed in the hydroponic system. Only S. nigrum was infected with PMTV through the roots, while S. nigrum together with C. album could be infected with PMTV by mechanical leaf inoculation. These results are in accordance with Jones & Harrison (1969, 1972) using virus-infested soil as inoculum for root infection. Jones & Harrison (1972) tested some of the same weed species as used in this study, namely L. communis, P. annua, P. avicutar, R. acetosella and V. tricolor, and obtained similar results. Foxe (1980) tested 26 weed species in PMTV-infested soil, including S. nigrum, S. arvensis and C. album, but did not observe any PMTV infection, even in S. nigrum. Jones & Harrison (1972) found zoosporangia in roots of C. album, M a t r i c a r i a matricarioides, S. nigrum, U. dioica, P. annua, L. c o m m u n i s and R. acetosetla but none in P. avicular and V. tricolor. In the present investigation no zoosporangia were found in P. annua but both P. avicular and V. tricolor hosted S.s.s. Foxe (1980) did Potato Research 45 (2002)

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BIRGITTEA.B. ANDERSEN,MOGENSNICOLAISENAND STEENLYKKENIELSEN find zoosporangia in S. nigrum and C. album, but not in roots ofS. arvensis. According to Karling (1968) Melhus et al. (1916), Ferdinandsen (1923), Janke (1965) and Wiirzer (1964) found S. nigrum as a host for S.s.s., but Wiirzer (1964) did not find L. communis and C. arvense as hosts for S.s.s., supporting the results of the present investigation. This variation in outcome of hosts for S.s.s. in these studies may be a result of differences in inoculum and experimental setup. In this study, the weed species were grown for 3 weeks in the hydroponic system before assessment for presence of zoosporangia. No root galls were observed in any of the weed species. This is not surprising as experiments with potato plants grown in the hydroponics had shown that root galls develops after 4 weeks (unpublished results). Our results show that PMTV has a very narrow natural host range, whereas S.s.s. has a much broader host range, among the 17 weed species. This suggests that the 17 weed species are unlikely to have been involved in the fast spread of PMTV in Denmark.

References Anonymous, 1986. Plant diseases, pest and weeds in Denmark 1986. 103rd annual report compiled by The Research Centre for Plant Protection. The Danish Research Service for Plant and Soil Science, Lyngby, 1988, pp. 39-41. Arif, M., L. Torrance & B. Reavy, 1995. Acquisition and transmission of potato mop-top furovirus by a culture of Spongospora subterranea f.sp. subterranea derived from a single cystosorus. Annals of Applied Biology 126: 493-503. Calvert, E.L., 1968. The reaction of potato varieties to potato mop-top virus. Record of Agricultural Research of the Ministry of Agriculture for Northern Ireland 17:31-40. Calvert, E.L. & B.D. Harrison, 1966. Potato mop-top, a soil-borne virus. Plant Pathology 15: 134-139. Clark, M.F. & N.A. Adams, 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for detection of plant viruses. Journal of General Virology 34: 475-483. Ferdinandsen, C., 1923. Ukrudtets betydning for plantesygdomme. Tidsskriftfor Landokonomi 6: 265-278. Foxe, M.J., 1980. An investigation of the distribution of potato mop-top virus in Country Donegal. Journal of Life Sciences, Royal Dublin Society 1: 149-155. Harrison, B.D. & R.A.C. Jones, 1970. Host range and some properties of potato mop-top virus. Annals of Applied Biology 65:393-402. Janke, C., 1965. Untersuchungen fiber den Wirtspflanzenkreis yon Spongospora subterrane a Wallr. Johns. Nachrichtenblatt fiir den Deutschen PJlanzenschutzdienst Berlin 19: 1-4. Jones, R.A.C. & B.D. Harrison, 1969. The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh. Annals of Applied Biology 63:1-17. Jones, R.A.C. & B.D. Harrison, 1972. Ecological studies on potato mop-top virus in Scotland. Annals' of Applied Biology 71 : 47-57. Karling, J.S., 1968. Powdery scab of potatoes and crook root of watercress. In: J.S. Karling (Ed.), The Plasmodiophorales, 2nd edition, Hafner Publishing Company, New York, pp. 180-192. M/ikgr~iinen, E., H. Rita, E. Teperi & J.P.T. Valkonen, 1994. Resistance to Spongospora subterranea in tuber-bearing and non tuber-bearing Solanum spp. Potato Research 37: 123-127. Melhus, I.E. & I.H. Vogel, 1918. Cabbage disease. Iowa Agricultural Experimental Circular 46:4 pp. 42


ALTERNATIVEHOSTS FOR POMOVIRUSAND ITS VECTOR Merz, U., 1989. Infectivity, inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system. Bulletin OEPP/EPPO Bulletin 19: 585-592. Nielsen, S.L. & J.P. Molgaard, 1997. Incidence, appearance and development of potato moptop furovirus-induced spraing in potato cultivars and the influence on yield, distributions in Denmark and detection of the virus in tubers by ELISA. Potato Research 40:101-110. Samson, R.G., T.C. Allen & J.L. Whitworth, 1993. Evaluation of direct tissue blotting to detect potato viruses. American Potato Journal 70: 257-265. Wfirzer, B., 1964. Ergfinzende Untersuchungen fiber den Pulverschof der Kartoffel und dessen Erreger Spongospora subterranea (Wallr.) Lagerh. Dissertation, Landwirtschaftliche Hochschule, Hohenheim., 104 pp. Zimmerman-Gries, S., 1972. Effects of mop-top virus on potato in Israel. Actas Congress Uniao Fitopatologia Mediterranea (Oliras) 3: 375-383.


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