Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 2, Num. 1, 1998, pp. 60-65

Angela Yordanova^1*, Dimitrinka Christova² and Todor Donev¹

¹National Bank for Industrial Microorganisms and Cell Cultures, Sofia 1113, P.O.Box 239, Bulgaria;
²Plant Protection Institute, 2230 Kostinbrod, Bulgaria

Code Number: cc98010

Summary

Investigations for conservation in liquid nitrogen of tospoviruses under the guise of infected leaf material and plant sap were performed. The protecting action of four cryoprotecting media was investigated . For reading the results an indicator test and ELISA were used. It had been registered that the specific biological properties and the virulent ability were fully preserved in the tospoviruses conserved with 10% dimethylsulphoxid and kept during three years in liquid nitrogen.

Introduction

In Bulgaria the "bronzing" on tobacco had been observed in 1952 by Ivantcheva - Gabrovska in the region of Gotse Deltchev and Sandanski [5]. Kovatchevski described, in the same year, two forms of action on tomatoes - a strong and a weak necrotic one [6]. The cause was the tomato spotted wilt virus (TSWV).

The TSWV possesses isometric particles with a diameter of 70 - 90 nm. The virions are covered with a membrane and contain lipids. The virus has a wide range of hosts and is spread in the temperate and subtropical regions of the world. This is one of the mostly inconstant plant viruses [4].

For a long time the TSWV was concerned to be the single representative of the monotype virus group of genus Tospovirus. Today, besides the TSWV, this genus includes also the impatiens necrotic spot virus (INSV) [3]. Some authors referred to the tospoviruses the tomato chlorotic spot virus (TCSV), the groundnut ringspot virus (GRSV) and groundnut but necrosis virus (GBNV), too [2, 10].

Because of the definitely expressed differences in the bronzing symptoms since the time of its observation in Bulgaria, as well as a result of its great economic importance, an investigation on the tospovirus dissemination in the country was started. The collected virus isolates were brought to identification, which required their preservation for a longer period of time. The variants of freeze-drying, successfully used for the conservation of other plant viruses in the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) collection, gave insufficient results when lyophilizing TSWV, which quickly lost its infectivity. This necessitated the application of other preservation methods.

The aim of this investigation was to develop reliable variants for long-term-storage of tospoviruses by the use of cryopreservation in liquid nitrogen.

Materials and Methods

Viruses and virus isolates. Isolates from tomatoes, pepper, tobacco and flower cultures, cultivated in greenhouse and in the open, were included in the study (Table 1). The virus isolates were transferred from diseased plants to tobacco (Nicotiana rustica or N. tabacum cv. 1146). The TCSV, GRSV and INSV were used as referent viruses. The INSV was maintained only on Impatiens sp.

Cryoprotectors. 10% glycerine, 10% dimethylsulphoxide (DMSO), 2% polyvinylpyrolidone - 10000 (PVP), 10% sorbitole + 3.6% dextran 40000 were used. The protecting media were prepared in 0.1 M potassium-sodium phosphate buffer, pH 7.2, containing 0.2% sodium sulphite and 0.2% ascorbic acid.

Freezing in liquid nitrogen (LN₂). 1/ Leaves - the infected leaves were cut and then 0.4 g were placed in cryotubes, containing 1.5 ml of protecting medium; 2/ sap - the infected leaves were homogenised with the above mentioned buffer in a ratio 1:2 (w/v). The resulting sap was centrifuged at 10000 rpm for 15 min. The supernatant was mixed with an equal volume of double concentrated protector and then 1.5 ml of this material was poured in cryotubes. All the operations were performed on ice (0°C).

The cryotubes were equilibrated at 4°C, as for the sap containing samples the duration was 1 h, while for the leaves containing ones it was 20 h. The freezing was accomplished with a speed of 0.5 - 0.6°C.min^-1 until a temperature -20°C had been achieved. Then the samples were transferred to liquid nitrogen and stored there.

The melting of the viruses was performed on water bath at 30°C.

Indicator test. The infected tissue from diseased plants was homogenised in 0.1 M potassium-sodium phosphate buffer, pH 7.2, containing 0.2% ascorbic acid and 0.2% sodium sulphite, in a ratio 1:1 (w/v). The diagnostic plants were inoculated and in 10 - 15 days the symptoms were read. Twelve plant species belonging to the families Solanaceae, Chenopodiaceae and Amarantaceae, were used.

After the cryopreservation of the viruses, the diagnostic plants were again inoculated, and the leaves containing samples had been preliminary ground. The symptoms were also read in 10 - 15 days. If there had been a lack of visible symptoms, a blind passage was performed.

Serological tests. Two modifications of ELISA were used for the serological identification of the isolates.

DAS - ELISA was accomplished according to the prescription of the firm "Loewe", Germany, respectively for TSWV, TCSV, GRSV and INRV.

TAS - ELISA was performed according to the following scheme: 1/ polyclonal antibody for TSWV (BR - 01) - 1:1000 in 0.05 M carbonate buffer, pH 9.6; 2/ antigen (plant sap) - 1:100 in PBS-T and 0.5% sodium sulphite; 3/ monoclonal antibody 4F2+[10] - 1:2000 in PBS-T, 2% PVP and 0.2% egg albumin; 4/ alkaline-phosphatase antimouse conjugate - 1:5000 in PBS-T and 2% bovine serum albumin. The extinction was read at 405 nm.

Results and Discussion

The initial tentative investigations had been performed with isolate 1To-94. Two variants of liquid nitrogen freezing were used - leaf material and plant sap, as four cryoprotectors had been applied (Table 2). The infectivity of the samples was tested 24 hours and 3 years after the freezing.

According to the data, the leaf variants had been better conserved. The virus was preserved in this form when protected with glycerine, DMSO and sorbitol/dextran. The last had been routinely used in the NBIMCC for the plant viruses lyophilization [11]. The glycerine, DMSO and PVP had been among the most frequently used protectors for low-temperature preservation of biological materials [1, 8]. PVP did not exhibit a protecting effect during our experiments. Other authors had also successfully applied glycerine, when freezing plant viruses [7, 9].

The results, achieved with glycerine and DMSO were comparable, but when few-times repetition had been performed, a better protecting effect of DMSO was observed. This cryoprotector had been chosen for the further investigations on the tospoviruses preservation.

Nine virus isolates were studied (Table 1). The characteristics, detected on the initial plants, varied too much between the different botanical species, but the chlorotic and necrotic spots and rings did predominate. It had been determined, by the use of TAS - ELISA that all the Bulgarian isolates belonged to the TSWV (Table 1).

The nine quoted TSWV isolates, as well as the referent TCSV, GRSV and INSV viruses were conserved in liquid nitrogen. The variant of leaves protected with 10% DMSO was used. All the viruses included in the investigation did keep their infectivity. The biological properties were compared before and after the freezing. The symptoms, induced by the Bulgarian isolates and the referent viruses on the diagnostic plants, did also characterise with a great variety, but were typical for the tospoviruses. Local symptoms were observed on Petunia hybrida, Chenopodium album, Ch. amaranticolor, Ch. foetidum and Ch. quinoa. In Datura stramonium, Gomphrena globosa, N. benthamiana, N. clevelandii, N. glutinosa, N. rustica and N. tabacum cv. 1146 the infection was local and systemic.

By testing on tomatoes, it had been established that the tomato isolates were extremely virulent for this culture. As concerning the tobacco isolates, 10HK-95 from North Bulgaria was quite more virulent for the tomatoes, when compared with the 4GD-95, that originated from South Bulgaria (Table1). The first one formed distinctly exhibited necrotic spots on the leaves and necrosis on the stem, while the second caused mosaic and chlorotic rings.

These specific biological properties of the virus isolates and the referent viruses are preserved after a three-year-period of conservation in liquid nitrogen. No changes, concerning the virulent ability, are observed.

After the revitalisation of the frozen in liquid nitrogen viruses, the samples were also tested with DAS-ELISA (Fig. 1). The individual representatives of genus Tospovirus distinctly differentiated. The belonging of the Bulgarian isolates to the TSWV was confirmed.

The results that have been achieved, show that the studied viruses, protected with DMSO - 10%, completely preserve their infectivity and biological characteristics after a freezing at minus 196°C.

This gives us a reason for applying the reported methodology in our routine work for liquid nitrogen conservation and preservation of tospoviruses.

Acknowledgements

The monoclonal antibody 4F2+ as well as the referent strains of TCSV, GRSV and INSV had been kindly granted to us by Prof. G. Adam from the Hamburg University, Germany, and we were very grateful to him.

References

1. Belous, A. M., M. I. Shrago, N. S. Pushkar, 1979. Cryoconservants, Kiev: Naukova dumka, 25 - 46. (In Russian)
2. De Avila, A. C., 1992. Diversity of Tospoviruses, PhD Thesis, Wageningen: Agr. Univ., Wageningen, The Netherlands.
3. De Avila, A. C., P. De Haan, E. W. Kitajima, R. de O. Kormelink, r. resende, R. W. Goldbach, D. Peters, 1992. J. Phytopathol., 134, 133.
4. Ie, T. S., 1970. CMI/AAB Description of Plant Viruses, No. 39.
5. Ivantcheva-Gabrovska, T., 1959. Scientific studies of the PPI, 2, 7-32. (In Bulgarian)
6. Kovatchevski, I., M. Markov, 1961. News of the PPI, 1, 5-24. (In Bulgarian)
7. McKinney, H.H., G. Silber, 1968. Methods of preservation and storage of plant viruses. In: Methods in virology, K. Maramorosch and H. Koprowski (eds.), vol. IV, New York: Academic Press, 491-501.
8. Pushkar, N. S., M. I. Shrago, A. M. Belous, U. V. Kalugin, 1978. Cryoprotectors, Kiev: Naukova dumka, 32-132. (In Russian)
9. Rochow, W. F., J.W. Blizzard, I. Muller, H.E. Warerworth, 1976. Phytopathology, 66, 534-536.
10. Roggero, P., G. Adam, R.G. Milne, V. Lisa, 1996. Eur. J. Plant Pathology, 102, 563-568.
11. Yordanova, A., E. Stoimenova, T. Donev, 1996. Biotechnol. Techniques, 10 (12), 977-982.

Copyright 1998 - National Bank for Industrial Microorganisms and Cell Cultures - Bulgaria

The following images related to this document are available:

Photo images