the effect of heat treatment and 'in vitro' - Annals of RSCB

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infections: apple (Campbell,M.V,1964; ... apple rootstock. (Larsen,E.C.,1974), strawberry (Posnette,A.F., 1959) and plum ..... Health certification of rosaceous.

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THE EFFECT OF HEAT TREATMENT AND ‘IN VITRO‘ CHEMOTHERAPY MEDIATED BY 2-THIOURACIL ON PLUM POX VIRUS (PPV) CONTENT IN MERISTEM REGENERATED PLUM PLANTS Z. Jakab-Ilyefalvi1, D. Pamfil1, Doina Clapa2, A. Fira2 1

UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA , DEPARTMENT OF BIOTECHNOLOGY, ROMANIA 2 FRUIT RESEARCH AND DEVELOPMENT STATION CLUJ-NAPOCA, ROMANIA

Summary The objective of this study was to test and evaluate the possibility of obtaining PPV free plants from infected plum plants ( Prunus domestica,L.cultivars ‘Iulia’ and ’Jubileu 50’) using a two step protocol using heat treatment in step one and 2 Thiouracil sysnthetic analog of guanosine with antiviral activity applying in step two. DAS-ELISA serological tests revealed that 2 Thiouracil has a small viral content reducing feature against PPV. Data shows that from a total of 15 plants a number of 5 plants could be regenerated (33.33 %) at cv. ’Iulia’ at 25 mg/L and 3 plants (20.00 %) at 35 mg/L concentration. Observations concluded that this retroviral has an effect of phytotoxicity. At a concentration of 35 mg/L the 2-Thiouracil induced a slight chlorosis and decreased shoot growth. Better results have been obtained at a decreased concentration of 25 mg/L 2-Thiouracil.Results showed that the viral content decreased from an average PPV content of 0, 850 OD to an average level of 0,350 OD, registering just 4 virus free plants (av.value of 0.115 OD). At `Iulia` cultivar results showed a multiplication index between 1:0.55-1:1.25 ‚ shoot lenght 0.85-1.15 cm, lateral shoot lenght 0.55-0.75 cm whereas at ` Jubileu 50` multiplication index was between 1:0.55-1:1.00 , lateral shoot lenght 0.350.65 mm. Keywords: meristem culture, plum pox virus, 2- Thiouracil, heat treatment.

[email protected] singular or in bunches or agregates in cases of high viral concentrations(Nemeth,1986). The main protein product of the plum pox virus has a dimension of 355 kDa.

Introduction Sharka disease caused by Plum pox virus is considered one of the most devastating viral diseases in stone fruits, especially in plum all over the world, causing high economical losses (20-90%), marketing depreciations. Plum pox virus is a member of the Potyviridae family, genus Potyvirus. The viral genome is a positive(+) oriented monocatene RNA helix having a dimension of 10 kb with a single segment and at the extremity is located a poly A tail (Krizbay, 2004; Jakab & Pamfil,2011). The viral particles are filamentous, having an average lenght of 720-900 nm and a diameter of 11-15 nm (Jakab, et al. 2011).The viral particles have a filamentous, flexuous exterior shape with a clear modal length, there are organized

Fig.1 Filimentous PPV virions at plum cultivar (scale bar=200 nm;Jakab et al.2011)

The serological proprieties of the virus are due to the capsidal protein (CP) , there are numerous viral strains ( PPVD,PPV-M,PPV-Elam, PPV –Rec e) (Briciu & Pamfil, 2011;Pamfil,1997).The viral 101

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horticultural species, especially Rosaceae fruit trees (Spiegel,1995;Hansen,A.J. et al.1983) which are more sensible to viral infections: apple (Campbell,M.V,1964; Postman,1995), raspberry (Converse,R.H.1966; Chambers,J.,1961), cherry (Snir,1985;Deogratias,J.M.,1989; Roux,N.,1987), peach (Kunkel,L.O.,1936), apple rootstock (Larsen,E.C.,1974), strawberry (Posnette,A.F., 1959) and plum (Janeckova,1993; Jakab et al.c) ,2011;Kouboris,2007;Vescan et al. 2011, Vertesy,1981;Polak,2009; Manganaris,et al. b) 2003; Paunovic et al.,2007;Diedzic,2008;Hauptmanova,2011) ,pear (Cieslinska;1999) prunus species (Cieslinska;2007), citrus (Greno,1990), almond (Rizqi,2001), peach (Stein,1991).

diagram of the virus is R/1:3,0-3,5/56:E/E:S/* Thermal inactivation point (T=10 min.) is 50-75 °C, longevity in cellular sap is 1-50 days, final dilution 10-1-10-6. Viral simptoms on the leaves are mosaic concentric zones , decoloration of the leaves, and the deformation of fruits (Nemet,M.,1986; Jakab,et al. d., 2011). Obtaining healthy initial planting material which is to be further used for building up vegetatively propagated certified stocks (Manganaris, 2003; Iqbal & Khan, 2010; Jakab et al.c., 2011) is the most important step in the setting up of a viable protocol in the establishment of plum orchards. Different methods are used in order to achieve a high value biological planting material: thermotherapy (Fridlund, 1989; Horvath,1999), chemotherapy, (Gabova,1995;Gella,1998;Mancino,1984), meristem culture and different combinations of these techniques (Howell,2001; Jakab et al.c), 2011; Hansen,1988; Knapp,1995). There have been elaborated with success, different meristem culture approaches and protocols regarding the various micropropagation phases (Quak, F., 1977; Hansen, A.J., 1989; Wang, P.J., 2006, Laimer, 1999, 2005). Several reports exists in agronomy and horticultural research about pathogen elimination systems experimented and mediated by individual chemotherapy using purine and pyrimidine compounds (Kamlesh,1981) like ribavirin (Jakab et al. 2010;Vescan et al. 2011; Permessur & Saumtally,2001;Manganaris,2003),thiouraci l (Francki,1962; Kuhn,1977) functioning as antagonists in the replication process of the viral core genome at several agricultural species:tobacco (Bawden,F.C.,1954),turnip (Commoner et al., 1952) and horticultural species: tomato (Milinko,1959), chrysanthemum (Ram,2005), potato (Conrad,1991), citrus (Sanjeev,2007). Individual heat treatment reports exists using a pre-established heat treatment procedure also in agronomical species and

Material and methods Thermotherapy Potted and properly rooted ‘in vitro’ plum plants - ‘Iulia’ and ‘Jubileu 50’ cultivars- were used for thermotherapy, primary tested for positive PPV status. Two sets of 15-15 plants according to the studied cultivars were introduced in the Snijders Scientific climate chamber of University of Agricultural Sciences and Veterinary Medicine Cluj, Faculty of Agriculture, and incubated at 250umol m-2s-1 light condition with increasing temperature from 28 oC to 39 oC. Two separate experiments were effectuated, the first heat therapy experiment had a duration of 15 days setting a 39 oC temperature at the Imago 3000 alphanumerical keypad , and the second experiment had a duration of 22 days at 37 oC. In the preliminary thermotherapy phase the plants were acclimated step by step (1 oC/day) until the set temperature was achieved. Humidification varied between 32-75 % rH. ‘In vitro’ meristem culture and chemotherapy. After completing the thermotherapy program the plum plants were transported to the Micropropagation laboratory where classic pre-sterilization and sterilization was effectuated (NaOCl 0,75 and 0,1 % for 5 min) with several rinsing phases with distilled water. 102

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irradiance of 55 umol m-2s-1 from cool white fluorescent lamps. Virus detection. Double antibody sandwich ELISA ( DAS-ELISA) was performed on the shoots derived from meristem tip explants(Clark,M.F et al., 1976). DAS-ELISA was employed using polyclonal antiserum for PPV detection (standard PPV detection, Kit-BIOREBA). The concentration of the coating immunoglobulin IgG was 1ug/ml and 0.5 ug/ml for PPV. Plant extracts ( leaves and shoots) were prepared by tissue homogenization in phosphate buffered saline –Tween containing PVP ( polyvinylpyrolidone). Each sample was tested twice and optical densities ( OD`s) were recorded at 405 nm with an ELISA plate reader 1 h after the substrate was added. Samples were considered ‘positive’ when OD values were more that twice the mean of the healthy control.

Meristem tip explants( apical dome with 2 leaf primordia-0,8 mm) were excised under an Olympus stereomicroscope. For explants culture the standard Murashige & Skoog (1962) plant medium, was used, supplemented with 30 % w/v sucrose and 0, 7 % w/v plant agar, ph adjusted to 5.8 before media autoclaving. For chemotherapy 2-Thiouracil antiretroviral compound was used, using two sets of concentration, 25 mg/L and 35 mg/L, the compound was first diluted in DMSO solvent and filter sterilized with Millipore filter after media autoclavation. The explants were cultured onto the MS medium and those with expanded shoots that were produced from meristem tip explants formed multiple shoots in subcultures. Shoots were cut from these subcultures were either used for virus testing or transferred to medium for future rooting. The cultures were maintained at 23 oC with a photoperiod of 16h and li. ng

Fig.1 ’In vivo’ thermotherapy of ’Iulia’ and ’Jubileu 50’plum cultivars Fig.2 Detail of vitro’shoot grown thermotherapy

’in in

conditions –Initial thermotherapy phase Fig.3 Intermediate thermotherapy phase Fig.4 Final thermotherapy Phase

Fig.5 ’In vitro’ cultured shoot apical meristems Fig.6 ’In vitro’ cultured shoot apical meristems

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Fig.7 Micromultiplication phase, ’ in vitro’ chemotherapy Culture media: MS+0.5 mg/L BAP Chemotherapy: 2-Thiouracil subjected to the first thermoterapy experiment ( T1=39 °C; 15 days) had a lower survival rate between 33.33-40.00 %, compared wit the second experiment (T1=37 °C; 22 days), where survival rate was higher 60-60,67 %.

Results and discussions Thermotherapy . Potted , branched , robust , regenerated ’in vitro’ plum plants ‚ ’Iulia’ and ’Jubileu 50’ cultivars infected with Plum pox virus ( PPV) were used for the heat treatment prior tested by DASELISA serological test. The plum plants

In the first 10 days the plants grew vigorous but gradiently at the final period of the treatment the shoot and leaves senesced due to the high temperature rates. Meristem tip explants ( of lenght 0,7-1.0) derived from the vegetative buds from the thermal treated plum plants (infected with PPV). Explant contamination had a lower value (6.25-7.14 %) but the necrosis after establishment had a higher value (56.2557.14 %) due to the high temperatures applied in the previous experiment and probable due to phenolic oxidation. A small percent of explants formed callus and these cultures were discarded from the

Cultivar Iulia Jubileu Total

Number of plants subjected to thermotheraphy 39 ˚ C 37 ˚ C 15 15 15 15 30 30

Duration of thermotherapy (days) 39 ˚ C 37 ˚ C 15 22 15 22

experiment. Meristem culture. A lower percent value of regenerated shoot apical meristems (SAM’s) showed viable characteristics, were green and turned to active morphological development phase. After 1 month of meristem culture the survived explants grew satisfactorily and could be subjected to subcultivation and multiplication. Meristem tips that were established succesfully and were subcultivated produced expanded healthy shoots and presented an average multiplication index of 1:2,17 ( cv. ’Iulia’) and 1:1,65 (cv ’ Jubileu’) . Temperature applied in thermotherapy Th. 1 Th.2 39 ˚ C 37 ˚ C 39 ˚ C 37 ˚ C

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Survival rate [no.] after thermotherapy 39 ˚ C 37 ˚ C 6 10 5 9 11 19

Survival rate [%] after thermotherapy 39 ˚ C 37 ˚ C 40.00 66.67 33.33 60.00 36.67 63.33

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Table 1. Multiplication parameters of cv. 'Iulia' and ' Jubileu 50' before chemotherapy with 2 Thiouracil Type of explant 'Julia' 'Jubileu 50'

Multiplication index 1:2,17 1:1,65

Length of axial shoot(cm) 1,39±0,31 1,26±0,37

Biometrical measurements results showed that the length of the axial shoot was 1.39 cm ± 0.31 at cv ’Iulia’ and 1,26 ± 0.37 at cv. ’Jubileu 50’ showing healthy tissues whitout of signs of senescence, viral symtoms of decoloration. Lenght of lateral shoots presented values between 0.46 ± 0.23 cm at cv.’ Jubileu 50’ and 0, 67 ±0,30 cm at cultivar ’Iulia’.The multiplied shoots were uniform in growth , with nodular callus at the base of the cuttings.Chlorosis effects were observed at the third subcultivation phase, but the multiplication index remained the same.

Length of lateral shoots (cm) 0,67±0,30 0,46±0,23

After the firs DAS-ELISA serological diagnostics, pozitive tested PPV plants were subjected to chemotherapy and the healthy plantlets were kept for further multiplication. The applied antiretroviral compound 2 Thiouracil is a synthetic analog to guanosine. We hardly found scientific literature available in aplying this antiretroviral compound . This compound is only soluble in DMSO , KOH and NaOH. We used to sets of concentrations of this compound: 25 mg/L and 35 mg/L at the plum cultivars ’ Iulia’ and ’Jubileu 50’

Table 2- `In vitro` regeneration through meristem culture of plum SAM `s after heat treatment Origin of explants

Number of cultured explants No.

Iulia Jubileu 50

%

Contamin ed explants No . %

Necrosis after establishme nt No.

16

100

1

6.25

9

14

100

1

7.14

8

105

% 56.2 5 57.1 4

Callus formation

Regenerated plantlets

No.

%

No.

%

1

6.25

5

31.25

1

7.14

4

28.57

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Data shows that from a total of 15 plants ( 3 repetitions of 5 plants) a number of 5 plants could be regenerated (33.33 %) at cv. ’Iulia’ at 25 mg/L and 3 plants (20.00 %) at 35 mg/L concentration. Observations concluded that this retroviral has a powerfull effect of phytotoxicity. At a concentration of 25 mg/L the 2 Thiouracil induced a slight chlorosis and decreased shoot growth was observed. Although generally the plantlets remained viable with firm nodular callus, good multiplication rate and no severe signs of phytotoxicity was observed. Increased signs of phytotoxicity was observed however at the increased 2 Thiouracil 35 mg/L concentration. Shoot growth

decreased massively , leaf chlorosis and necrosis was observed at leaf and shoot level. In ’Jubileu 50’ plum cultivar the regenerated plantlets percent varied between 13.33-20.00 % , showing evident phytotoxicity signs. Cultivar ’Jubileu 50’ is less adapted to biochemical stress. Multiplication indexes showed lower values when compared with those of regenerated after heat treatment. At `Iulia` cultivar results showed a multiplication index of 1:0.55-1.25 ‚ shoot lenght 0.85-1.15 cm, lateral shoot lenght 0.55-0.75 cm whereas at ` Jubileu 50` multiplication index was between 1:0.551.00 , lateral shoot lenght 0.35-0.65 mm.

Table 2. Multiplication parameters of cv. ' Iulia' and 'Jubileu 50' upon 2 Thiouracil treatment in subculture I and II Number of cultured explants Origin of explant Prunus domestica cv 'Iulia' 2 Thiouracil 25 mg / L 2 Thiouracil 35 mg / L Prunus domestica cv 'Jubileu 50' 2 Thiouracil 25 mg / L 2 Thiouracil 35 mg / L

Multiplication index

Length of axial shoot[cm] Means ± SD

Length of lateral shoots [cm] Means ± SD

Sub. I

Sub. II

Sub. I

Sub. II

Sub. I

Sub. II

15

1:1,25

1:0,85

1,15±0,22

1,05±0,22

0,75±0,22

0,45±0,22

15

1:0,55

1:0,35

0,85±0,23

1,05±0,23

0,55±0,23

0,35±0,23

15

1:1,00

1:1,00

1,05±0,22

0,85±0,22

1,05±0,22

1.00±0,22

15

1:0,55

1:0,55

0,55±0,23

0,65±0,23

1,05±0,23

0,65±0,23

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cowpea and observed that in a single treatment after 48 hours the virus titre decreased 90 % of SBMV particles.Francki, (1962) concluded that 2-TU suprresses de production of infective TYMV nucleoprotein in chinese cabbage and the TU analog induces a a very marked increase in the production of virus protein shells containing no viral RNA. The total amount of virus protein per leaf ( empty protein shell plus protein in nucleoprotein) may exceed the amount the proteins in control leaves.Peter Conrad (1991) succeded the elimination of Potato virus S at 100 varieties of potato with the aim ot aplying 2TU.Experiments carried out at lilium for elimination of Lilium symptomless virus(LSV) concluded that at succesiv concentrations of 20,40,80 µM 2TU the survival percent at scales was 2% and at bulbs 47% (Jin Ha Woo, 2004). Sherin (2010) observed a direct decrease of virus content for Potato spindle tube viroid , at a level of 20 mg/L survival rate of plants was 63.3-70 % and elimination success was 63 %.At and increased level of 50 mg/L 2-TU concentration. In our researches we observed that the efficiency in the elimination of plant viruses of this antiretroviral compound 2 Thiouracil greatly depends on concentration, type of host plant genetic material and type of virus. Higher concentration than 35 mg/L cannot be aplied in the pathogen elimination systems in the case of 2Thiouracil chemotherapy in meristem culture at plum, because it couses growth inhibition,chlorosis and necrosis at this level of concentration.It is recomended that 2 Thiouracil if aplicable needs to be used between 10-25 mg/L concentration. Results showed that it has been achieved an average regeneration rate of 28-31 % after chemotherapy with 2 TU although just a small number of virus free plants have been obtained ( 4 plants) which showed a healty physiologica status ( 0.115 OD ) regarding the PPV content.

Virus testing DAS-ELISA serological tests revealed that 2 Thiouracil has a slight viral load reducing feature against PPV although just a very litle percent of virus free plants were recorded and it is not entirely clear if this is directly the result of the meristem culture technique influence or the direct effect of 2 Thiouracil. Results show that the viral content decreased from an average PPV content of 0, 850 OD to an average level of 0,350 OD, registering 4 virus free plants (av.value of 0.115 OD).

Efficiency of 2-Thiouracil (2-TU) in the pathogen elimination systemns although it was tested on several species:tobaco,chrisanthemum,rosa,turnip,t omato,potato,lily,carnation, sunflower, chinese cabbage ,apple, there is a lack of researches in the application of 2TU applied at rosaceaus plants especially at Prunus species. The antiretroviral compound 2-Thiouracil although partly causes phytotoxicity and growth inhibition, it could be used in chemotherapy programs with adequate setting of concentration. Ram,R. et al. (2005) concluded that 2-TU applied at chrisanthemum in concentration 0,04 g/dm3 induces 26,7 % virus free plants if this chemotherapy step is followed by a heat therapy program of 38 oC for 30 days. Kuhn,et al.(1977) experimented 2TU in 107

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and in vitro culture. CAN.J.PLANT PATH. , 11:337-342. Ewa, D. (2008). Elimination of Prunus necrotic ring spot virus ( PNRSV) from plum `Earlyblue`shoots through thermotherapy in vitro. Journal of Fruit and Ornamental Plant Research , 101-109. Francki, R. (1962). Some effects of 2 Thiouracil on the multiplication of turnip yellow mosaic virus. Virology,Elsevier Inc , 367-380. Francki, R. (1962). The inhibition of plant virus multiplication in two host species by 2 Thiouracil. Virology, Elsevier, Inc. , 17(1):18. Fridlund, P. (1989). Thermotherapy. Virus and Virus like Diseases of Pome Fruits and Simulating Noninfectious Disorders.Washington State University Coop Ext. Publication SP0003 , 284-295. Gabova, R. (1995). Chemotherapy treatment of Prunus ssp genotypes infected by Plum pox virus. Plant Science ,Sofia , 32:16-18. Gella, R., & Errea, P. (1998). Application of in vitro therapy for Ilarvirus elimination in three Prunus species. J. PHYTOPATH. , 146:445449. Greno, V., M., C., L., N., & N., D. (1990). Effect of antiviral chemicals on the development and virus content of citrus buds cultured in vitro. Scientia Horticulturae , 45(1-2):75-87. Hansen, A. (1989). Antiviral chemicals for plant disease control. CRC Critical reviews in Plant Sciences , 8: 45-88. Hansen, A. (1988). Applied virology research vol.1 ( E. Kurstak, R.G. Marusyk, F.A. Murphy, M.H.V. Van Regenmortel, Eds.) . Plenum Publishing Corporation , 285-299. Hauptmanova, A., & Polak, J. (2011). The elimination of Plum pox virus in plum cv. Bluefree and apricot cv. Hanita by chemotherapy of in vitro cultures . Hort. Sci. Prague , 49-53. Horvath, J., & Gaborjanyi, R. (1999). Növényvírusok és virológiai vizsgálati módszerek. Mezogazda k. . Howell, W., Eastwell, K., & T.S.C., L. (2001). Heat treatment, chemotherapy and hydroponic culture for obtaining virus free trees of sweet cherry. Acta Horticulturae , 550:455-458. Iqbal M., Khan M. A. (2010) Management of Cotton leaf Curl Virus by planting time and plant spacing. AAB Bioflux, 2(1):25-33.

Acknowledments This work has benefited from financial support through the 2010 POSDRU 89/1.5/S/52432 project, “ ORGANIZING THE NATIONAL INTEREST POSTDOCTORAL SCHOOL OF APPLIED BIOTECHNOLOGIES WITH ON ROMANIAN BIOECONOMY” project co-financed by the European Social Fund through the Sectorial Operational Programe Human Resources Development 20072013.

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meristem culture. Plant Virology;Proceedings of the 9 th Conference of the Czechoslovak Plant Virologist;Brno . Vescan, L., D. Pamfil, Zagrai, I., V.Berindean, I., Clapa, D., Ciuzan, O., et al. (2011). In vitro techniques for Plum pox virus elimination from two infected Romanian Plum Cultivars. Bulletin UASMV,Animal Sciences and Biotechnologies , 68(1-2):389395. Wample, G.I., M., R., & Howell, W. (1998). Heat treatment of perennialplants to eliminate phytoplasmas, viruses and viroids while maintaining plant survival.Am. Phyt,S.,USA , 332-345. Wang, L., Wang, G., Hong, N., Tang, R., Deng, X., & Zhang, H. (2006). Effect of thermotherapy on elimination of apple stem grooving virus and apple chlorothic leaf spot virus for in vitro-cultured pear shoot tips. HORT-SCIENCE , 41 (3):729-732.

improved in vitro shoot-tip grafting. Acta Hortic. , 550:447-454. Sanjeev, S., Balvinder, S., Gita, R., Zaidi, A.A., Vipin, H., Avinash, N., et al. (2007). Production of indian citrus ringspot virus free kinnow plants employing chemotherapy coupled with shoot tip grafting. In vitro Cell Dev.Biol.-Plant , 43:254-259. Snir, I., & Stein, A. (1985). In vitro detection and elimination of prunus necrotic ringspot virus in sweet cherry ( Prunus avium). Riv. Ortifloro Fruit, IT. , 69:191-194. Spiegel, S., & Stein, A. (1995). In vitro thermotherapy of Rosaceae fruit trees. Acta Horticulturae , 386:419-420. Stein, A., Spiegel, S., Faingersh, G., & Levy, S. (1991). Responses of micropropagated peach cultivars to thermotherapy for elimination of Prunus necrotic ringspot virus. Ann.Appl.Biol. , 119:265-271. Vertesy, J. (1981). Elimination of Plum pox virus (Prunus domestica,L.) rootstocks by

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