+Bioline International Official Site (site up-dated regularly)

African Crop Science Journal, Vol. 9. No. 3, pp. 517-525

BIOLOGICAL CHARACTERISTICS OF TOMATO MILD MOTTLE VIRUS A POTYVIRUS ISOLATED FROM TOMATO AND THORN APPLE IN ETHIOPIA

YAYNU HISKIAS¹, D. - E. LESEMANN and H.J. VETTEN

Federal Biological Research Centre for Agriculture and Forestry, Institute for Biochemistry and Plant Virology, Messeweg 11-12, 38104 Braunschweig, Germany
¹Address for correspondence: Ethiopian Agricultural Research Organisation (EARO), P. O. Box 2003, Addis Ababa, Ethiopia

Received 31 August, 2000
Accepted 3 February, 2001

Code Number: cs01071

ABSTRACT

Two isolates of the virus 246/94 and 277/94, acquired from thorn apple (Datura stramonium) and tomato (Lycopersicon lycopersicum (L.) Karst. Ex. Farw., syn. esculentum), respectively, were characterised biologically and serologically and compared with a local isolate of Potato virus Y (PVY) isolated from tomato and other potyviruses and isolates infecting vegetables. Both isolates of TMMV infected only 16 of 28 plant species inoculated mechanically and induced indistinguishable symptoms. The most susceptible hosts were D. metel, D. stramonium and Nicotiana glutinosa L. However, these isolates differed from the PVY isolate by infecting Datura spp. and Solanum demissum L., while the PVY isolate infected Chenopodium quinoa Wild and Capsicum annuum L. Isolate 277/94 was transmitted non-persistently by the aphid, Myzus persicae Sulz. from diseased tomato to virus-free D. stramonium, D. metel L., N. glutinosa and tomato plants and from these back to virus-free test plants of each species. Purified particles of isolate 277/94 contained a single protein species with a molecular weight of 39 kDa. Furthermore, in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) antiserum to isolate 277/94 reacted strongly with a Yemeni isolate of TMMV (Y90/7), but did not react with any other potyvirus. This clearly shows that the Yemeni and Ethiopian isolates of TMMV are similar serologically.

Key Words: Aphid transmission, host range, potyvirus, serology, tomato mild mottle virus

RÉSUMÉ

Datura stramonium et Nicandra physalodes Gaertn en Ethiopie. Deux formes du virus 246/94 et 277/94, ont ete obtenue respectivement partir du Datura stramonium et de tomates (Lycopersicon lycopersicum (L.) Karst. Ex. Farw., syn. esculentum) infectees. Leurs compositions biologique et sérologique ont été identifiées et comparées avec: d'autres potyvirus, un virus présent dans les pommes de terre locales Y (PVY) et isolé à partir des tomates, et d'autres formes de virus présents dans des légumes contaminés. Les deux formes du virus TMMV ont contaminé 16 des 28 plants inoculés mécaniquement, provoquant des symptômes similaires. Les plants où le virus s'est cependant le mieux développé sont: le D. metel, le D. stramonium et le Nicotiana glutinosa L. Cependant, ces deux formes different du virus PVY dans la mesure où ils ont contaminé le Datura spp. et le Solanum demissum L, alors que le PVY a contaminé le Chepodium quinoa Wild et le Capsicum annuum L. Le virus 277/94 a été transmis de manière non persistante par l' Aphid, Myzus persicae Sulz. Des tomates contaminées vers du D. stramonium, D. metel L., N. glutinosa et des plants de tomate saints, puis de ces plants infectés vers d'autres plans tests saints de chaque espèce. Des particules purifiées du virus (277/94) contiennent une seule espèce de protéine avec une molécule pesont 39 kDa. De plus, une enzyme sandwich d'anti-corps immunosorbent assay (DAS-ELISA) antiserum permettant d'isoler le 277/94, a réagi fortement avec la forme Yéménite du virus TMMV (Y90/7). Ceci démontre clairement que les formes Yéménite et éthiopienne du virus TMMV sont très proches d'un point de vue sérologique.

Mots Clés: Transmission des aphides, gamme des hôtes, le virus de la tomate milde mottle

INTRODUCTION

About 30 plant viruses belonging to 11 different taxonomic groups infect tomato in different countries of the world among which members of the Potyviridae predominate (Jones et al., 1991). Among the potyviruses, Potato virus Y (PVY) (Jones et al., 1991), Tobacco etch virus (TEV) (Harold, 1970; Jones et al., 1991), Pepper veinal mottle virus (PVMV) (Ladipo and Roberts, 1977; Jones et al., 1991) and Peru tomato virus (PTV) (Fernandez-Northcote and Fulton, 1980; Jones et al., 1991) have been reported from tomato and other vegetables, while Eggplant green mosaic virus (Ladipo, 1976) and Tomato mild mottle virus (TMMV) infect only tomato (Walkey, 1992; Walkey et al., 1994). Tomato mild mottle virus was described from Yemen (Walkey, 1992), a country adjacent to Ethiopia and has now been found to be the dominant and widespread pathogen of tomato in Ethiopia both in single infections and mixed infections with PVY (Yaynu et al., 1999). In addition to tomato (Lycopersicon esculentum) the virus was also found infecting thorn apple (D. stramonium) and N. physalodes weeds growing in association with tomato crops, in adjacent open fields, along river banks and lake sides. Walkey et al. (1994) compared the serological relationship of TMMV isolated from tomato in Yemen with only few other potyviruses that infect tomato and pepper and observed no cross-reactions between TMMV and the potyviruses.

The widespread occurrence of TMMV in tomato in Ethiopia as reported recently (Yaynu et al., 1999) suggests that there may be many sources of infection and an efficient transmission mechanism of the virus. Thus, it is necessary to identify the sources of infection and the transmission mechanisms in the affected areas and in greenhouse. This paper presents and discusses the host range, aphid vector transmission and some biological and serological characteristics of TMMV isolates from tomato (L. esculentum) and thorn apple (Datura stramonium) in Ethiopia. Additionally, the Ethiopian TMMV isolates are compared with a Yemeni isolate of the virus and other potyviruses from vegetables in Ethiopia and other countries.

MATERIALS AND METHODS

Virus isolates and host range. Isolates of TMMV; 246/94 from D. stramonium and 277/94 from tomato were collected in 1994 in the rainy and dry seasons at Koka and Zwai in the Rift Valley of Ethiopia, respectively (Yaynu et al., 1999). Isolate PVY-356/94 was collected from tomato from Melkasa also in the Rift Valley. All TMMV isolates were maintained in Nicotiana glutinosa and PVY-356/94 in N. tabacum var. White Burley by sub-culturing every 30 days. Infected leaves were also stored frozen at -20⁰C to prevent possible contamination in the greenhouse. Extracts from two-week-old systemically infected leaves were prepared as described earlier (Yaynu et al., 1999). The resulting extracts were inoculated each onto 4 carborundum-dusted test plants of the 16 plant species listed in Table 1 and also to Gomphrena globosa L. (Amaranthaceae), Chenopodium amaranticolor Coste & Reynier, C. foetidum Schrad., C. foliosum Aschers (Chenopodiaceae), Cucumis sativus L. (Cucurbitaceae), Zea mays L. (Gramineae), Phaseolus vulgaris L., Vicia faba L. (Fabaceae), N. hesperis L., N. miersii Remy Nr. 33, and N. sylvestris Speg & Comes (Solanaceae) under greenhouse conditions. All plants were inoculated at the 2-3 leaf-stage. The experiment was repeated once and only species infected in both experiments were included in Table 1. In addition to visual observation, the presence of virus in the plants was confirmed by testing both inoculated and non-inoculated leaves by ELISA (Clark and Adams, 1977).

Aphid transmission. Isolate 277/94 was transmitted by Myzus persicae from infected tomato plants cv. Linda to virus-free test plants of tomato cv. Linda, D. stramonium, D. metel, N. glutinosa and from these back to virus-free test plants of each species. Apterous M. persicae were fasted for 3-4 hr, allowed an acquisition access period of 5-10 min on 277/94-infected plants and transferred to virus-free test plants of each species using 30 aphids per plant. The experiment was repeated once and results recorded. Aphids were killed after an inoculation access period of two days. Equivalent acquisition accesses to virus-free plants of all species were included as controls.

Virion length determination. For particle length measurements, virions were trapped by immunosorbent electron microscopy (ISEM) from extracts of 277/94-infected plants. The lengths of 100 particles of isolate 277/94 were measured at a magnification of 50,000x directly in a Zeiss EM906 electron microscope using an on-line attached image analysing system (Digivion, SIS, Muenster, Germany) with histogram production and normal length calculation.

Cytopathology of infected tissues. Cyto-pathological effects induced by isolate 277/94 in N. tabacum cv. Xanthi-NC were studied as described by Koenig and Lesemann (1985). Freshly harvested leaves were cut into pieces, prefixed in 2.5% glutaraldehyde, fixed in 0.5% osmium tetroxide and washed overnight in 1% uranyl acetate, pH 4.3. Leaf tissues were dehydrated by sequential periods in increasing acetone concentrations for 90 min and in a 1:1 (v/v) mixture of acetone and Epon-812 for 3 h. Leaf pieces were embedded in Epon-812, polymerised at 60⁰C for 48 h and ultrathin sections were made using an ultramicrotome (LKB Ultratome III). The ultrathin sections were viewed and photographed in a Zeiss EM 906 transmission electron microscope.

Virus purification. Isolate 277/94 was passed through D. stramonium to exclude from PVY and propagated in N. glutinosa. Leaves showing mosaic symptoms were harvested 10-12 days after inoculation and stored frozen until use. Frozen leaves were purified as described previously (Siriwong et al., 1995; Ravi et al., 1997; Yaynu and Vetten, 2000), without the sugar density gradients. The purity and concentration of virus preparations were examined by electron microscopy and yields of purified viruses estimated spectrophoto-metrically (UV scan at 200 - 400 nm) as shown in Noordam (1973).

Immunological and serological studies. Antiserum to isolate 277/94 was produced in a rabbit by weekly intramuscular injections of 200-400 mg of purified antigen emulsified in an equal volume of Freund's incomplete adjuvant for six weeks. Serological relationship of the isolate with other viruses was studied in DAS-ELISA in homologous and heterologous combinations using a total of 10 antisera as described by Clark and Adams (1977).

Coat protein size determination. Molecular weight (MW) of the coat proteins (CP) of purified isolate 277/94 and other local potyvirus isolates (374/94 and 430/94) from hot pepper were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4% stacking gel on a 12% resolving gel and the buffer systems of Laemmli (1970). Gels were run in a vertical electrophoresis apparatus (Mighty small II, Hoeffer Scientific Instruments, San Francisco). Purified virus preparations were denatured by boiling at 95⁰C for 5 min. Amounts equivalent to 0.3, 0.25, 0.20 and 0.25μg of purified preparations of isolate 277/94, 374/94 and 430/94, respectively, were loaded into the slots and electrophoresed first at 80V until the bromphenol blue reached the resolving gel and continued at 120V until they reached the bottom of the gel.

RESULTS

Experimental host range. The reactions of 16 test plant species to mechanical inoculations with PVY-356/94, TMMV-246/94 and TMMV-277/94 are listed in Table 1. The two TMMV isolates infected 16 solanaceous species on which symptoms were indistinguishable. All species reacted with systemic symptoms except N. occidentalis, which developed local lesions within 3 days of mechanical inoculation. The species highly susceptible to both isolates were D. stramonium, D. metel, L. lycopersicum cv. Linda, N. physalodes, N. benthamiana, N. clevelandii, N. glutinosa and N. tabacum vars. White Burley and Xanthi-NC of which the last two species were used for virus multiplication and purification. These species developed various conspicuous systemic symptoms 6-8 days after inoculation as compared to two weeks for those inoculated by the PVY-356/94. Moreover, the majority of the species gave highly specific DAS-ELISA readings with the TMMV-277/94 antiserum. Although many plant species from different families were included in the host range study, the Ethiopian isolates of TMMV only infected species in the Solanaceae, but not C. annuum. Also N. miersii, N. sylvestris and N. hesperis were further solanaceous species infected by the Ethiopian isolates of TMMV. As shown in Table 1 the TMMV isolates differed from the PVY isolate by infecting Datura spp. and S. demissum, while the PVY - 356/94 infected C. quinoa and hot pepper, which appeared to be immune to the TMMV isolates.

Aphid transmission. Isolate TMMV-277/94 was transmitted non-persistently using all combi-nations of source and recipient host plant species, except from N. glutinosa to D. stramonium and vice versa (Table 2). Symptoms in N. glutinosa and the two Datura spp. appeared 8-10 days after aphid inoculation, whereas in tomato symptoms became visible after two weeks. The non-persistent mode of transmission by M. persicae is characteristic of members of the genus Potyvirus (Hollings and Brunt, 1981).

Particle morphology. Filamentous particles of c. 12 nm in diameter with a modal length of 700 nm were observed consistently in leaf extracts infected with isolate TMMV-277/94. This was unexpectedly short for a potyvirus, whose normal length ranges from 680 to 900 nm (Hollings and Brunt, 1981). This, however, is consistent with a value of 719 nm for isolate TMMV Y90/7 from Yemen (Walkey et al., 1994).

Cytopathological effects. As shown in Figure 1 cytoplasmic cylindrical inclusions (CI) were rare in TMMV-277/94-infected tissue, which may be due to the extremely low particle concentration observed in crude extracts. Moreover, the few CIs found in the 277/94-infected cells were incompletely developed such that a clear assignment to one of the five CI types could not be made. However, it seemed that CIs belonged to either type II or type IV as described previously (Walkey et al., 1994).

Properties of purified virus preparation. Virus yields of only 2-4 mg per kg of leaves were obtained for isolate TMMV-277/94, indicating a very low concentration of this isolate in the source plant. Purified preparation of the isolate still contained a considerable amount of host protein assumed to be fraction I (Fig. 2). The purity of the purified preparation was also examined by spectrophotometry and SDS-PAGE, the latter of which confirmed the presence of impurities in the virion prepation as shown in Figure 2. The purified virus had a UV absorption spectrum typical of the nucleoprotein of potyviruses with a maximum at 260 nm, a minimum at about 246 and a slight shoulder at 290 nm. The A260 nm/A280 nm ratio of the isolate was 1.06, which is lower than the range typical for potyviruses (Noordam, 1973). This might have been due to the presence of impurities.

Serological relationship as determined by DAS-ELISA. The Ethiopian isolate TMMV-277/94 was analysed in homologous and heterologous combinations by DAS-ELISA using homologous antiserum and antisera to Yemeni isolate, Y90/97 as well as eight antisera to other potyviruses infecting tomato and pepper locally and other countries. The antisera to the Yemeni and Ethiopian isolates of TMMV reacted not only in homologous and heterologous combinations, but also with four other Ethiopian potyvirus isolates from tomato, D. stramonium and N. physalodes, indicating that they are all serologically closely related and can be regarded as TMMV isolates (Table 3). None of the two TMMV antisera reacted with any of the eight other potyviruses and their isolates, and no DAS-ELISA reactions were observed when the antisera to different potyviruses and isolates were tested against the six TMMV isolates.

Capsid protein size. The SDS-PAGE analysis of purified preparation of isolate 277/94 revealed that the isolate contained one major protein band with M_r of 39.0 (Fig. 3 lane b). However, the preparation also yielded, in addition to the major band, two minor bands of c. 36 and c. 50 kDa. The former appeared to be a proteolytically degraded form of the viral CP (Shukla et al., 1992), while the latter was regarded as a protein co-sedimented with virions during purification (Fig. 2). Isolate TMMV-277/94 had a CP size of c. 39 kDa which is in agreement with a CP size of 42 kDa previously reported for a Yemeni isolate of the virus (Walkey et al., 1994).

DISCUSSION

The incidence and severity of TMMV in tomato and associated solanaceous weeds in many regions of Ethiopia were reported earlier (Yaynu et al., 1999), but its infection route is not fully understood and must be determined in order to devise appropriate control measures. Sixteen Solanaceous species were identified as hosts of both TMMV isolates under greenhouse conditions. Most of the hosts developed systemic infections, except N. occidentalis, which produced local lesions to both TMMV isolates. This species can be used as an assay host and for infectivity test with this virus. Most of the hosts included in this experiment are wild or annual weed species that grow fast and abundantly together with tomato in both rainfed and irrigated fields, along river banks and lake sides throughout the year.

The natural vector(s) of TMMV is/are not yet known. However, under experimental conditions the virus was transmitted from different infected plant species to test plants by M. persicae in a non-persistent manner, typical of potyviruses (Hollings and Brunt, 1981). This aphid species is known to be polyphagous (Minks and Harrewijn, 1987) and is anholocyclic in the tropics including Ethiopia, allowing rapid reproduction. Moreover, non-persistent transmission by aphids provides fast virus spread from infected to healthy plants in the field. The virus presumably over-winters on hosts such as D. stramonium, and N. physalodes (Yaynu et al., 1999) growing continuously along lake sides and river banks. To break the disease cycle, over-wintering hosts growing together with tomato should be removed so that vectors do not transmit the virus.

The formation of cytoplasmic cylindrical inclusions (CIs), also referred to as pinwheels is the major criterion for the assignment of a new isolate to the Potyviridae (Edwardson, 1974; Edwardson and Christie, 1996; Shukla et al., 1992). The CIs induced by TMMV-277/94 were very rarely found, which is probably due to extremely low particle concentrations as observed in the crude extracts.

Different serological methods have been used to estimate the extent of cross-reactivity among virus species of different groups (Clark and Adams, 1977; Koenig, 1978). The DAS-ELISA method is highly specific and has been used to differentiate between strains of the same virus, while indirect ELISA has been applied to reveal distant relationships (Koenig, 1978; Koenig and Paul, 1982). An antiserum was produced against isolate 277/94 of TMMV and its relationship with potyviruses tested in homologous and heterologous combinations in DAS-ELISA. There was no serological difference between the Ethiopian and the Yemeni isolate of TMMV, while no cross reaction was observed between these and the other potyviruses. This shows that TMMV is a serologically distinct virus as also shown by Walkey et al. (1994).

Isolate 277/94 was conspicuous in having a CP size of c. 39 kDa, which is largely in agreement with CP size of 42 kDa previously determined for the Yemeni isolate (Walkey et al., 1994). Although SDS-PAGE revealed some proteolytic degradation of TMMV CP, the apparent discrepancy between the size determinations is probably due to different experimental conditions but not due to proteolytic degradation of TMMV-277/94 CP. Moreover, the CP of the Yemeni and Ethiopian isolates were indistinguishable in size when analysed by electro-blot immunoassay (Vetten, personal observation). Another minor band clearly observed above the major band could be due to impurities, co-sedimented during purification.

REFERENCES

Clark, M.F. and Adams, N. 1977. Characteristics of the microtitre plate method of enzyme-linked immunosorbent assay (ELISA) for the detection of plant viruses. Journal of General Virology 34:475-483.
Edwardson, J.R. 1974. Some properties of the potato virus Y-group. Florida Agricultural Experiment Station Monograph Series No. 4, 398 pp.
Edwardson, J.R. and Christie, R.G. 1996. Cylindrical inclusions. University of Florida
Agricultural Experimental Station Bulletin 874, 79 pp.
Fernandez-Northcote, E.N. and Fulton, R. W. 1980. Detection and characterisation of Peru tomato strains infecting pepper and tomato in Peru. Phytopathology 70:315-320.
Harold, F. 1970. Tobacco etch virus in Venezuela. Plant Disease Reporter 54:334-345.
Hollings, M. and Brunt, A.A. 1981. Potyviruses. In: Handbook of Plant Virus Infections and Comparative Diagnosis.
Kurstak (Ed.). Elsevier, Amsterdam. pp. 731-807.
Jones, J.B., Jones, J.P., Stall, R.E. and Zitter, T.A. 1991. Compendium of Tomato Diseases. American Phytopathological Society Press, St. Paul, Mn. 132 pp.
Koenig, R. 1978. ELISA in the study of homologous and heterologous reactions of plant viruses. Journal of General Virology 49:309-318.
Koenig, R. and Paul, H.L. 1982. Detection and differentiation of plant viruses by various ELISA procedures. Acta Horticulturae 127:147-158.
Koenig, R. and Lesemann, D.-E. 1985. Plant viruses in German rivers and lakes. Phytopathologische Zeitschrift 112:105-116.
Ladipo, J.L. 1976. A green mosaic disease of eggplant (Solanum melogena) in Nigeria. Plant Disease Reporter 60:1068-1072.
Ladipo, J.L. and Roberts, I.M. 1977. Pepper veinal mottle virus associated with a streak disease of tomato in Nigeria. Annals of Applied Biology 87:133-138
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bactriophages T4. Nature 227: 680-685.
Minks, A.K. and Harrewijn, P. 1987. Aphids: Their Biology, Natural Enemies and Control. Vol. 2A. Elsevier Science Publishers. B.V. Amsterdam. 700 pp.
Noordam, D. 1973. Identification of Plant Viruses, Methods and Experiments. Centre for Agricultural Publishing and Documentation, Wageningen, Netherlands. 207 pp.
Ravi, K.S., Joseph, J., Nakaraju, S., Krishna P. S., Reddy, H.R. and Savithri, H.S. 1997. Characteristics of a pepper vein banding virus from chilli pepper in India. Plant Disease 81: 673-676.
Shukla, D.D., Lauricella, R. and Ward, C.D. 1992. Serology of potyviruses. Current problems and some solutions. In: Potyvirus Taxonomy. Barnett, O.W. (Ed.). Archives of Virology 5: 57-69.
Siriwong, P., Kittipakorn, K. and Ikegami, M. 1995. Characterization of chilli vein banding
mottle virus isolated from pepper in Thailand. Plant Pathology 44:718-727.
Walkey, D.G.A. 1992. Plant virus of Yemen and associated areas. London, UK: Overseas Development Administration. 168 pp.
Walkey, D.G.A., Spence, N.J., Clay, C.M. and Miller, A. 1994. A Potyvirus isolated from Solanaceous hosts. Plant Pathology 43:931-937.
Yaynu Hiskias, Lesemann, D.-E. and Vetten, H.J. 1999. Occurrence, distribution and economic importance of potyviruses infecting hot pepper and tomato in Ethiopia. Journal of Phytopathology 147:5-11.
Yaynu Hiskias and Vetten, H.J. 2000. Biological properties of potyviruses from hot pepper (Capsicum annuum L.) in Ethiopia. Ethiopian Journal of Pest Management 4:29-39.

The following images related to this document are available:

Photo images