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THERMOTHERAPY AND ELECTROTHERAPY OF POTATO TUBERS INFECTED WITH POTATO VIRUS Y – PVY Conference Paper · October 2011
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СЪЮЗ НА УЧЕНИТЕ В БЪЛГАРИЯ – СМОЛЯН Юбилейна национална научна конференция с международно участиe: “ЧОВЕКЪТ И ВСЕЛЕНАТА” 6-8 октомври 2011 г., Смолян, БЪЛГАРИЯ ----------------------------------------------------------------------------------------------
UNION OF SCIENTISTS IN BULGARIA Jubilee National Scientific Conference with International Participation “ THE MAN AND THE UNIVERSE” th th October, 6 -8 , 2011, Smolyan, BULAGRIA
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THERMOTHERAPY AND ELECTROTHERAPY OF POTATO TUBERS INFECTED WITH POTATO VIRUS Y – PVY Nikolay Petrov, Valya Lyubenova Plant Protection Institute, Kostinbrod, Bulgaria
ТЕРМОТЕРАПИЯ И ЕЛЕКТРОТЕРАПИЯ НА КАРТОФЕНИ КЛУБЕНИ ЗАРАЗЕНИ С КАРТОФЕНИЯ ВИРУС Y – PVY Николай Петров, Валя Любенова Институт за защита на растенията, Костинброд
Резюме: Род Потивирус обхваща почти една трета от известните досега растителни вируси. Много от тях причиняват значителни загуби на продукция по различни култури от сем. Chenopodiaceae, Commelinaceae и Solanaceae, от които с найголямо икономическо значение са картофите, тютюна и зеленчуковите култури. С найголямо икономическо значение за България е картофеният вирус Y – PVY. Целта ни е да елиминираме вируса от картофените клубени чрез използването на различни схеми на третиране, базирани на температура и електрическо напрежение. В нашето изследване ние използваме инфектирани с PVY картофени клубени от два сорта Аринда и Агрия. Схемата на третиране се разделя на три части: третиране на клубените само с гореща вода, третиране само с 15 mA 3V електричество в буфер и комбинация от двете. Резултатите ни показват, че третирането с гореща вода и намаляване на силата и продължителността на електрическо напрежение са най-добрият начин за постигане на свободни от вирус картофени клубени и запазване на кълняемостта на картофите. Ключови думи: PVY, термотерапия, електротерапия, картофени клубени
INTRODUCTION Potato (Solanum tuberosum L.) is one of the most important plants in human nutrition. Seed tuber quality is an extremely important factor for potato yield. Since it is a vegetatively-propagated plant, fungal, bacterial and, particularly viral disease agents are easily transmitted through the tubers [31]. Viral diseases are easily transmitted via infected tubers from one generation to the next and from one region to another, causing considerable yield and quality losses. They are, for the most part, responsible for degeneration, characterized by a decrease in vigor, productivity, and resistance to diseases of potato cultivars after successive cultivation from the same lot of tubers [26, 27]. Many viruses naturally infect potatoes wherever they are grown [6, 13]. Few viruses are transmitted in true potato seed [6, 15], whereas most, if not all, are transmitted in vegetatively propagated tubers [6, 13]. Virus diseases are the major limiting factor of potato production and cause its deterioration [1, 2, 10, 12, 14, 18, 21, 23]. Potato virus Y-PVY is one of the most damaging, widespread viruses, which cause diseases in potato. PVY is the type species of the genus Potyvirus, in the family Potyviridae [17]. It occurs as long, flexuous particles measuring 730nm x 11nm, containing single-stranded, positive polarity RNA [9]. High PVY level can cause stand loss, reduced yields, undersized tubers and reduced quality [7, 11]. Symptoms induced by
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PVY vary from an almost imperceptible mosaic up to severe necroses and premature death of plants, depending on cultivar and viral strain [28, 29]. Over the past twenty years, PVY has become an increasingly serious constraint to seed potato production in the world [8, 19]. Thus efforts to control PVY are essential when producing potatoes for market or seed. [3,4,5]. Conventional control methods to viral diseases which are based on the control of their viral vectors are generally economically costly. Therefore, cultivation of disease-free seed tubers is considered the most effective method of controlling potato viruses [22]. The efficiency of conventional methods of virus eradication from plants including meristem culture and thermotherapy is low and they are not successful for all viral infections. These methods have certain limitations due to time length requirement and less efficiency in producing virus-free plants [22]. Electrotherapy was reported to be successful in eliminating PVX [20], PVY and PLRV [24]. AIM Decreasing yield losses and quality damages caused by Potato virus Y using different schemes of electrotherapy and thermotherapy. MATERIAL AND METHODS 1. Plant material Potato tubers from two cultivars Agria and Arinda were used. For each experiment we used 10 tubers from each cultivar. Germs from each tuber were tested with ELISA and PCR, so selection was only for infected tubers with potato virus strain PVYO. They were treated directly in different schemes of electrotherapy and thermotherapy. After the treatment each tuber was sown in separate pot and when growing the plant was tested for the presence or absence of PVY. 2. Detection of the virus
All tubers were tested for PVY after germination and growing the plats with DASELISA using sap from homogenized potato leaves( or from dead tubers were used tissues from the tuber) as an antigen Double Antibody Sandwich- DAS ELISA was performed following a protocol described by Clark and Adams (1997). Micro titer ELISA plate wells were coated with PVY IgG antiserum diluted in carbonate buffer (pH 9.6) according to the supplier‘s (LOEWE Biochemica GmbH Sauerlach, Germany) specifications. IgG used in the experiment was diluted 1:200 (v/v) in the 0.05 M carbonate buffer. Plates were incubated for 4 h at 37°C, followed by 3, 5-minute washing steps with PBS-T buffer and then loading with homogenized in coating buffer with 1% PVP and albumin(BSA) plant extracts. After that plates were incubated at 4°C overnight. After washing off the crude plant extract, virus was detected by PVY antibodies conjugated with alkaline phosphatase and diluted in conjugate buffer according to the supplier‘s specifications in incubation step for 4h at 37°C. p-nitro phenyl phosphate diluted in diethanolamin buffer (1mg ml -1, pH 9.8) is a substrate for the alkaline phosphatase enzyme reaction which run on room temperature and after coloring is stopped with 3N NaON. Optical density at 405 nm was measured by Multifunctional detector DTX 880 (Beckman, USA). Tissue samples from healthy and infected plants were used as negative and positive controls. Positive results are these that exceed two time optical density of the negative control (positive result > 2x 0.180 OD405 = 0.360). Therefore, tested samples, with OD405 value more than 0.360, were considered positive for PVY infection. 3. Treatment schemes a/ Thermotherapy Hot-water treatment of dormant tubers from PVY infected plants of the two cultivars were treated in five cycles (for each cycle and duration time for 30 infected tubers) at 50°C, 52°C, 55°C, 60°C, 70°C with three different duration times each- 10 min, 20min and 30 min. For each scheme there is negative control of healthy potato tuber to measure germination rate compared to infected and treated tubers and one untreated infected tuber as positive control for each cultivar. b/ Electrotherapy Electrotherapy treatment of the tubers from PVY infected plants of the two cultivars were treated at seven different levels of electric currents (for each level and time courses for 30 infected tubers) at 5 mA, 10 mA, 15 mA, 20 mA, 25 mA, 30 mA, 35 mA with five time courses each- 3 min, 5 min, 12 min, 20 min and 30 min in big electrophoresis tank. Electric currents were supplied by an
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electrophoresis power supply and potato tubers were sunk in 1M NaCl water solution buffer. For each scheme there is negative control of healthy potato tuber to measure germination rate compared to infected and treated tubers and one untreated infected tuber as positive control for each cultivar. c/ Combination of thermotherapy and electrotherapy We tested all possible combinations between successful cycles and duration times in hotwater thermotherapy and successful levels of electric currents and time courses in electrotherapy to achieve better results with higher germination rates of potato tubers. First we apply electrotherapy and after that thermotherapy cycles. Efficiency of all schemes of treatment was estimated according to the following formula: efficiency of therapy = virus elimination percent x percent of tuber germination. RESULTS AND DISCUSSION Effects of thermotherapy treatment on eliminating PVY Hot-water treatment in the temperature interval 50°C to 55 °C and the chosen duration times for all treated potato tubers from both cultivars infected with PVY showed no positive effect of freeing the infected tuber from the virus. All the tubers remain infected with PVY. There were no differences in results from the tested potato cultivars Agria and Arinda. Hot-water treatment of the potato tubers in 60 °C changed the picture and we received our firs success in elimination the PVYO potato strain from both cultivars (tabl.1). Elimination of the virus from the tubers was 30% but germination of the tubers was reduced to 25 %. At 70 °C we received 40 % virus free tubers but unfortunately the germination rate was 0% and all potato tubers was dead. All infected controls of untreated tubers have 100 % germination. So, further experiments with thermotherapy alone were meaningless. With these results we confirmed that heat treatment alone is not successful in freeing potato tubers from PVY and other potato viruses [16, 25, 30]. Effect of electrotherapy treatment on eliminating PVY Plants were grown successfully from almost all electrotherapy treated potato tubers from the cultivars Agria and Arinda. The electric currents of 5 mA and 10 mA for all the five time courses tested were unsuccessful for PVY virus elimination from potato tubers. Levels of electric current, more than 15mA, show different antiviral effect depending on the duration of treatment and genotype of the cultivar (tabl.2). The treatment of 25mA for 12 min was generally the most effective among the treatments in reducing the virus concentration of PVY up to 70% and reducing germination rate to 60 %. This treatment had the highest effect of virus elimination and comparatively lowest reduction of germination rate from cv. Agria followed by cv. Arinda. Germinated infected control potato tubers without electrotherapy treatment were tested positive for PVY after growing indicating that diminishing virus titer was due to electrotherapy. Similar effect of electrotherapy of potato plantlets (potato stem segments with 3-5 axillary buds) against PVY was demonstrated by Meybody [22]. They used 35mA for 10 min to eliminate PVY from potato stem segments. Compared to our results this level of electric current is too high for poato tubers from cv Agria and cv Arinda. Actually electric current more than 30mA for more than 10 min eliminate completely PVY from the tubers but there was no germination at all. That‘s why we reduced the electric current to not more than 25mA for 12 min. duration time more than 12 min eliminate the virus much more but reduces germination at the same time. Earlier attempts to eliminate PVY using 7mA resulted, merely, in decreased levels of virus titer in regenerated plants [32]. Combined scheme of therapy The best combination that we achieved combining electrotherapy followed immediately by thermotherapy was electric current 15 mA for 10 min and 55°C for 15 min in water bath. This scheme eliminate the virus at 93.3 % and reduce the germination rate at 60% of cv. Agria, and 90 % virus elimination and 73.6 germination rate of cv. Agria. Increasing the electric current in electrotherapy or temperatures in thermotherapy in combination schemes resulted in sharply decreased germination rate and death of potato tubers. CONCLUSION Electrotherapy and hot-water thermotherapy, compared to conventional virus elimination techniques, as meristem culture and hot-air treatment for long periods of time, are technically simple, faster and thus economically less costly. Tuber germination rate in electrotherapy and hotwater thermotherapy is not so high compared with other techniques, but the PVY virus elimination is much higher and much more effective than all other methods used up to now. Based on our
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results, these methods can be used as an effective technique for elimination potato viruses from potato tubers. Conditions for these therapies can differ for each virus strain-host-environment system. Therefore it is essential to determine optimal treatment conditions for a desired system. Further studies are required to optimize the system for better results for fully elimination the virus and to increase the germination rate of potato tubers. The choice of potato cultivar and the virus strain are critical factors together with the conditions of the electrotherapy and thermotherapy for achieving better results. REFERENCES: [1] Al-Shahwan, I.M.; Abdalla, O.A.; and Al-Saleh, M.A. (1998). Potato viruses in central Saudi Arabia. Journal-of- King-Saud-University,-Agricultural-Sciences. 1998, 10: 1, 45-53. [2] Arif, M.; Mughal,S.M.; Khalid, S.; and Hassan, S. (1995). Some biological physical and serological properties of potato leaf roll virus (PLRV) in Pakistan. Pakistan- Journal-of-Botany. 1995, 27: 1, 233-241. [3] Badarau, C., Chiru, S., Cojocaru, N., Ianosi, M., Chiru, N. 2010. Studies regarding the improvement of methods used for viruses identification in potato seed indexation. In: Potato agrophysiology. Proceedings of the International Symposium on Agronomy and Physiology of Potato, Nevsehir, Turkey, pp: 332-340 [4] Badarau, C., Cojocaru, N., Rusu, S., Ianosi, M., Petrusca, K. 2009. The effect of samples incubation on detection of PLRV and the influence of several extraction buffer‘s additives on the detection of potato viruses Y, A, X and S by ELISA technique. In: Proceeding of the 2nd International Symposium ―New Researches in Biotechnology‖, Series F , Biotechnology, Bucharest, 2009, pp : 9-17 [5] Badarau, C., Marculescu, A., Cojocaru, N., Rusu, S., Ianosi, M. 2010. Studies regarding the improvement of methods used for the potato‘s viruses identification. In: Bulletin Issue of International Conference on New Research in Food and Tourism Bioatlas, Journal of EcoAgroTurism, Transilvania University of Brasov Publisher Brasov, pp: 83-91 [6] Beemster, A. B. R., Rozendaal, A., 1972. Potato viruses: properties and symptoms. Pages 115-143 in: J. A. de Bokx, ed. Viruses of potatoes and seed-potato production. Centre for Agricultural publishing and documentation (PUDOC), Wageningen, The Netherlands. [7] Beemster, A., De Bokx, A. 1987. Survey of properties and symptoms. In: Viruses of potato and seed potato production, eds. J.A.de Bokx and J.P.H. van der Want, Wageningen, The Netherlands RUDOC, pp: 284-290; [8] Davis, A., Radcliff, E.,Schrage, W., Rgsdale, D. 2008. Vector and virus IPM for seed potato production. In: Insect pest management: Concepts, tactics, strategies and case studies, eds. Radcliffe E.B., Huchison W.D.,Cancelado R.E.,Cambridge, UK, Cambridge University Press, , pp:366-377 [9] De Bokx, J.A.; Huttinga, H. 1981Potato Virus Y. Descriptions of Plant Viruses, n.242 [10] Hamm, P.B. and Hane, D.C. (1999). Effects of seedborne potato leafroll virus on Russet Norkotah potato. Plant-Disease. 1999, 83: 12, 1122-1124. [11] Hane, D., Hamm, ,B. 1999. Effects of seedborne Potato virus Y infection in two potato cultivars expressing mild disease symptoms. Plant Disease, 83: 43-45 [12] Hord, M.J. and Rivera, C. (1998). Prevalence and geographic distribution of viruses PVX, PVY, PVA, PVM, PVS and PLRV in potato in Costa Rica. Agronomia-Costarricense. 1998, 22: 2, 137-143. 23. Jan, H. and Khan, S.B. (1995). Incidence and distribution of potato viruses in the Upper Kaghan Valley of Pakistan. Pakistan-Journal-of-Phytopathology. 1995, 7: 1, 13 16. [13] International potato center,1977. The potato: major diseases and nematodes. Centro Internacionalde la Papa, Lima, Peru. 68 pp. [14] Jan, H.; Khan, S.B.; and Mohammad, A. (1994). Occurrence and distribution of potato viruses in the Upper Kagham Valley of Pakistan. Sarhad-Journal-of- Agriculture. 1994, 10: 6, 691696. [15] Jones, R. A. C., Fribourg, C., 1977. Beetle, contact and potato true seed transmission of Andean potato latent virus. Ann. Appl. Biol. 86:123-128 [16] Kassanis, B. 1950. Heat inactivation of leaf roll virus in potato tubers. Ann. Appl. Biol. 37:339-341. [17] Kitajima, E.W.; De Avila, A.C.; Resende, R.O., 1997.Taxonomia de virus de plantas. Fitopatologia Brasileira, v.22, p.5-24 [18] Kurppa, A. (1983). Potato viruses in Finland and their identification. Journal-of-the
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Scientific-Agricultural- Society-of-Finland. 1983, 55: 3, 189-301. [19] Lorenzen, J., Meacham,T., Berger, P., Pat, J., Crosslin, J., Hamm, P., Kopp, H. 2006. Whole genome characterization of potato virus Y isolates collected in the western USE and their comparison to isolates from Europe and Canada., Archives of Virology, 151: 1055-1074 [20] Lozoya, H., Abello, F. and Garcia, G. 1996. Electrotherapy and Shoot Tip Culture Eliminate PVX in Potatoes. AM Potato J. 73: 149-154. [21] Mansour, A.N. (1999). Incidence of potato viruses in Jordan. Dirasat Agricultural Sciences. 1999, 26: 3, 313-319. [22] Meybody, D., Mozafari, J., Babaeiyan, N., Rahimian, H. 2011. Application of electrotherapy for the elimination of potato potyviruses. J. Agr. Sci. Tech. 13: 921-927 [23] Omer, A.D. and El-Hassan, S.M. (1992). Incidence of potato viruses and their effect on potato production in the Sudan. Crop-Protection. 1992, 11: 5, 477-479. [24] Pazhouhandeh, M. 2001. Establishment of in vitro Gene-bank for Virus-free Potato Germplasm. MSc Thesis, Tarbiat Modarres University. [25] Rozendaal, A. 1952. Demonstration of experiments with potato viruses. Pages 63-65 in: Pro. First conf. Potato virus diseases, Lisse – Wageningen, The Netherlands, 1951. [26] Sangar, R.B.S.; Agrawal, H.O.; Nagaich, B.B., 1988. Studies on the translocation of potato viruses X and Y in potatoes. Indian Phytopathology, v.41, p.327-331 [27] Silberschmidt, K., 1937. A degenerescência da batatinha. O Biológico, v.9, p.247-254 [28] Silberschmidt, K.; Kramer, M., 1942. O vírus Y, uma das principais causas da degenerescência da batatinha no Estado de São Paulo. O Biológico, v.8, p.39-46 [29] Souza Dias, J.A.C.; Iamauti, M.T. Doenças da batateira. In: Kimati, H.; Amorim, L; Bergamin Filho, A.; Camargo, L.E.A; Rezende, J.A.M. (Ed.). Manual de Fitopatologia. São Paulo: Agronômica Ceres, 1997. v.2, p.137-164. [30] Thomson, A., 1956. Heat treatment and tissue culture as a means of freeing potatoes from virus Y. Nature 177: 709. [31] Truta, A.A.C., 1997. Detecção simultânea de vírus em batata (Solanum tuberosum L.) por DAS-ELISA e determinação do material vegetal ideal a ser utilizado nos programas de indexação. Lavras: UFV, 59p. [32] Yoon, J., Wonseo, H., Mee Choi, Y. and Eun Park, Y. 2003. Ribavirin, Electric Current, and Shoot-tip Culture to Eliminate Several Potato Viruses. J. Plant Biotechnol. 5(2):101105.
Nikolay Manchev Petrov, Plant Protection Institute, 35 P. Volov Str., 2230 Kostinbrod, Bulgaria Tel.: +359 72168811; Fax. +359 72166062 e-mail:
[email protected] Valya Kostadinova Lyubenova Plant Protection Institute, 35 P. Volov Str., 2230 Kostinbrod, Bulgaria Tel.: +359 72168811; Fax. +359 72166062
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APPLICATION
(%)
Healthy
(%)
Dead tubers Infected
(%)
Healthy
(%)
Infected
tubers
Surviving Tubers Survival of
Germination/
Replicates
period (min)
Treatment
(°C)
of treatment
Temperature
Cultivar
Potato
Table 1 Effects of thermotherapy treatment against PVY infected tubers.
10
10
6
100
0
100
0
20
10
7
100
0
100
0
30
10
5
100
0
100
0
10
10
5
100
0
100
0
20
10
4
100
0
100
0
Agria and
30
10
5
100
0
100
0
the same
10
10
2
100
0
100
0
20
10
1
100
0
100
0
with
30
10
1
100
0
100
0
Arinda
10
10
4
75
25
66.8
33.2
20
10
4
75
25
66.8
33.2
30
10
2
50
50
62.5
37.5
10
10
0
0
0
60
40
20
10
0
0
0
60
40
30
10
0
0
0
60
40
50
52
results
55
60
70
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Table 2 Effects of electrotherapy treatments against PVY infected tubers Potato Cultivar
Electric current (mA)
Agria 5
10
15
20
Agria 25*
30
35 Arinda 5
10
15
Time courses (min) 3 5 12 20 30 3 5 12 20 30 3 5 12 20 30 3 5 12 20 30 3 5 12* 20 30 3 5 12 20 30 3 5 12 20 30 3 5 12 20 30 3 5 12 20 30 3 5 12 20 30
Replicates
Survival of tubers
Virus free tubers
Infected tubers
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 8 8 7 7 8 6 6 6 4 4 2 2 2 1 1 0 0 0 0 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 1 4 5 7 7 6 7 7 7 8 8 10 10 10 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 10 9 9 9 6 5 3 3 4 3 3 3 2 2 0 0 0 0 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
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20
25*
30
35
● 3 5 12 20 30 3 5 12* 20 30 3 5 12 20 30 3 5 12 20 30
●
ISBN: 978-954-397-025-4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
7 7 7 6 7 5 5 5 2 2 1 1 1 1 1 0 0 0 0 0
*Numbers in grey indicate the most effective combination
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