TOBAMOVIRUS STRAIN P101 ISOLATED FROM PEPPER IN BULGARIA

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1 TOBAMOVIRUS STRAIN P101 ISOLATED FROM PEPPER IN BULGARIA E. Stoimenova 1, A. Yordanova 2 Institute of Genetics, Sofia, Bulgaria 1 National Bank for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria 2 ABSTRACT The tobamovirus strain P101 was isolated from pepper growing in greenhouse in Bulgaria. It does not differ from the other tobamoviruses in virion morphology, stability in sap, preparations, dried and frozen leaves as well as in the thermal inactivation point. The ratio A 260 /A 280 of P101 purified preparation is 1.3 and it is higher than ones for other viruses from the group. The strain P101 has close electrophoretic mobility to paprika mild mottle virus (PaMMV, strain P11). When the homologous antiserim is used, P101 and other typical virus strains from the tobamovirus group arrange in the following series of serological relationship: P101 PaMMV > tobacco mild green mosaic virus >pepper mild mottle virus > tobacco mosaic virus > tomato mosaic virus. P101 and strain P11 of PaMMV are strongly related but not serologically identical. Both strains overcome the L1 resistance gene (pathotype P1) but at least 28 accessions of Capsicum chinense are resistant to P101 and susceptible to P11. The Bulgarian strain differs from P11 by the reaction of tomato and Nicotiana tabacum cv. Samsun N N. However, the established differences between both strains are not enough to propose P101 as a separate virus and it can be define as a strain of PaMMV. Introduction Tobamoviruses occur frequently in pepper grown in polyethylene and glasshouses and are relatively rare for the field pepper. Tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) cause severe losses mainly in susceptible cultivars of pepper. A group of tobamovirus strains is found infecting pepper cultivars resistant to TMV and ToMV. These strains are designated as pepper strain of TMV (8). The resistance against tobamoviruses is conferred by allelic series of genes with increasing effectiveness of the locus L: L+<L1<L1C<L2< L3<L4 (2). The L-mediated resistance of pepper plants is manifested as hypersensitive reaction (HR) and formation of necrotic local lesions (NLL). According to their ability of breaking the resistance conferred by the respective alleles of gene L, the tobamovirus strains are divided into Biotechnol. & Biotechnol. Eq. 19/2005/2 30 four pathotypes P0, P1, P1.2 and P The strains belonging to pathotype P0 induce HR in all resistant genotypes; pathotype P1 overcomes the L1-madiated resistance and elicits HR in pepper plants possessing L2, L3 and L4 genes; pathotype P1.2 breaks the resistance conferred by L1 and L2 genes but induces HR in L3 and L4 plants; pathotype P1.2.3 systemically infects plants with all resistance genes with the exception of L4 gene. Consequently, the strains could be arranged by their increasing ability to overcome resistance genes in the following series of pathotypes: P0<P1<P1.2<P1.2.3 (2). Nowadays the pepper strains are differentiated in two viruses: pepper mild mottle virus (PMMoV) and paprika mild mottle virus (PaMMV) (3, 4, 14). The first virus includes P1.2 and P1.2.3 and the second P1 pathotype strains. Virology, Supplement

2 The object of the present work was to determine the strain P101 belonging to a virus from the tobamovirus group, summarizing new and previous investigations on its properties. Materials and Methods Viruses and hosts. Strain P101 isolated from pepper was studied. The following viruses and strains were also used: TMV - U1; ToMV - GM-0; TMGMV (tobacco mild green mosaic virus) - U2; PaMMV P11, PMMoV - GP2 and GP3 (1, 4, 9, 12). Nicotiana tabacum cvs. Samsun, Samsun N N and Samsun NN; Capsicum annuum cvs. Albena and Tissana; C. frutescens cv.tabasco; C. chinense PI ; C. chacoense PI and Lycopersicon esculentum cv. Ideal were used as host plants. The strains were propagated as follows: strain P101 and P11 in C. annuum cv.tissana; GP2 and GP3 in C. frutescens cv.tabasco and C. chinense PI , respectively; GM-0 in tomato cv. Ideal; U1 and U2 in tobacco cvs. Samsun N N and Samsun, respectively. The tobamoviruses were purified according to Gooding and Hebbert (5). The seeds of the test plants were sown in pots with sterile soil and tobacco seedlings were transfered in sterile soil too. The tomato and pepper plants were infected in cotyledon - primary true leaf phase, while the tobacco - in 5 th - 6 th leaf phase. The plants were inoculated with purified preparations (0.05 mg/ml) and the symptoms were recorded 7, 14 and 28 days after the infection. Electrophoretic mobility of virus particles. It was determined by agarose electrophoresis (3). Rf = S/F was calculated, where S was the way the sample had passed, and F was the distance between the start and the front. The values from the three replications were recalculated in relation to Rf of U1, accepted as 100%, and the averages were taken. Immunodiffusion tests. Double radial diffusion was made in 0.7 % agarose gel in M veronal buffer, ph 8.6, containing 5 mm EDTA. Purified virus preparations in concentration 5 mg/ml and antiserum against P101 (dilution 1:2 1:4) were used. The antigen had been applied 30 hours before the antiserum. The precipitation lines were observed after incubation for 48 hours at room temperature. Counter immunoelectrophoresis was performed in 0.9 % agarose gel in 0.75 M veronal buffer, ph 8.6, at 150 V, 10 ºC for hours. The virus preparations in concentration 1 mg/ml and the antiserum against P101 diluted 1:3 1:10 were applied. In the immunodiffusion tests the gels were stained with Coomassie Brilliant Blue R-250 (15). Results and Discussion In 1979 it was found that more than 80 % of the glasshouse pepper cv. Bruinsma Wonder in village of Marten, Rousse region, were infected with a tobamovirus althow this cultivar possessed resistant gene (6). All the isolates were similar in their biological properties and were named as strain P101. Strain P101 did not differ from the other tobamoviruses in the virion morphology, stability in preparation, sap, dried or frozen leaves, as well as in the thermal inactivation point (6, 11, 12). The ratio A 260 /A 280 of P101 purified preparation is 1.3 (13). The established value was higher than the ones of other viruses from the group: TMV , ToMV , PMMoV and TMGMV (1). Our former results showed that P101 and PMMoV strains move too slowly in polyacrylamide gel and therefore the latter is not suitable for electrophoretic mobility determination in our experiments (13). Agarose gel provided more precise separation of the viruses (Fig. 1). The average Rf (%) values for the tested strains were: P ± 1.4 %, PaMMV 42.2 ± 0.8 %, TMGMV ± 0.3 %, PMMoV strains GP ± 7.5 % and GP ± 8.4 %, TMV 100 % and ToMV Virology, Supplement 31 Biotechnol. & Biotechnol. Eq. 19/2005/2

3 Fig. 1. Agarose electrophoresis of strain P101 and other tobamoviruses. Lines: ToMV GM-0 (1), TMV U1 (2), TMGMV U2 (3), PaMMV P11 (4), P101 (5), PMMoV GP3 (6), P101 (7) and TMV U1 (8) ± 1.0 % (Fig. 2). P101 moved slower than the other viruses with an exception of PaMMV and both strains had the closest Rf values. The serological relationship of strain P101 and other tobamoviruses was studied by means of counter immunoelectrophoresis. A homologous P101 antiserum was used. In immunoelectrophoretic analysis a very strong reaction was observed between P101 and the serum, so its precipitation lines disposed near the start and they did not encounter with the bands of the other viruses. The behavior of the strain P11 was similar. Some variants of electrophoresis were tested where the antigens and the antiserum were loaded at different time. The most convenient was the application of P101 and P11 one hour before the rest viruses and the antiserum. Strains P101 and P11 produced spur against TMV, ToMV, TMGMV and PMMoV (Fig. 3). The bands of P101 and P11 were quite similar but fusion was not observed even at multifold dilutions of the reagents. From the rest viruses U2 formed spur towards U1, GM-0 and GP3; GP3 to U1 and GM-0; U1 to GM-0 (Fig. 3). Double immunodiffusion was also performed. When the antigen concentrations were varied, a weak spur of P101 towards P11 was observed (Fig. 4). In this test the reaction of the both viruses was also much stronger in comparison to the other tobamoviruses which precipitation lines were weaker and unclear. Strain U2 formed spurs to TMV, ToMV and PMMoV. The obtained results show that when the homologous antiserum is used, the tobamovirus P101 and other typical virus strains from the group arrange in the following series of serological relationship: P101 > PaMMV > TMGMV > PMMoV > TMV > ToMV. The reaction of host range plants, after inoculation with P101, TMV, ToMV, TMGMV, PaMMV and PMMoV, was compared. (Table). The data of the infection of the pepper cultivars and wild species, possessing the respective allelic genes, showed that TMV, ToMV and TMGMV belonged to P0 pathotype, strain P101 and PaMMV - to P1, PMMoV - to P1.2 and P Infecting a large amount (84) of C. chinense accessions, Kostova discovered that 28 accessions are resistant to P101 and susceptible to PaMMV (7). The author supposes that a gene Lx, between L1 and L2, locates P101, but could not withstand P11. Consequently, according to the ability of overcoming the resistance genes strain P101 and other viruses could form a series: TMV = ToMV = TMGMV < P101 < PaMMV < PMMoV. Biotechnol. & Biotechnol. Eq. 19/2005/2 32 Virology, Supplement

4 Rf (%) U1 GM-0 U2 P101 P11 GP2 GP3 Fig. 2. Electrophoretic mobility (Rf) of strain P101 and typical tobamovirus strains: TMV U1, ToMV GM-0, TMGMV U2, PaMMV P11, PMMoV GP2 and GP3. Rf of TMV U1 is accepted as 100 %. U1-GM0 U2-GM0 GM0-GP2 GM0-GP3 GP2-GP3 U1-GP3 U2-GP3 P101-U1 U2-P101 P101-GP2 GP3- P101 P101-GM0 U1-U2 P11-U1 GM0-P11 P11-U2 GP3-P11 P11-P101 Fig. 3. Immunoelectrophoretic analysis of serological relationship of strain P101 and PaMMV strain P11 with other tobamoviruses: TMV U1, ToMV GM-0, TMGMV U2, PMMoV GR2 and GR3. In upper holes - P101 antiserum. The response of tobacco cv. Samsun NN to infection with P101 and other tobamoviruses was identical. NLL developed on the inoculated leaves. The NLL induced by P101 Virology, Supplement 33 Biotechnol. & Biotechnol. Eq. 19/2005/2

5 TABLE Comparison of symptoms* on host range plants induced by strain P101 and the main viruses from the tobamovirus group Host plants and resistance genes Strain P101 Viruses TMV ToMV TMGMV PaMMV PMMoV C. annuum cv. Albena L+/L+ M M M M M M C. annuum cv. Tisana L 1 /L 1 M NLL NLL NLL M M C. frutescens cv. Tabasco L 2 /L 2 NLL NLL NLL NLL NLL M C. chinense PI L 3 /L 3 NLL NLL NLL NLL NLL M/ NLL C. chacoense PI L 4 /L 4 NLL NLL NLL NLL NLL NLL L. esculentum cv. Ideal Tm+ /Tm+ SS M M N. tabacum cv.samsun SL M M M SL SL N. tabacum cv.samsun N N SL/NLL M NLL NLL NLL NLL N. tabacum cv.samsun NN NLL NLL NLL NLL NLL NLL *Symptoms: M mosaic, NLL - necrotic local lesions, SS - systemic symptomless infection, 0 - no infection, SL -symptomless infection in inoculated leaf;, / - or. P11 GP3 P101 U2 U1 GM0 Fig. 4. Double immunodiffusion test of strain P101 and other tobamoviruses: TMV U1, ToMV GM-0, TMGMV U2, PaMMV P11 and PMMoV GP3. In central hole - P101 antiserum. inoculation emerged 48 h later being smaller than those caused by the other viruses. ToMV, TMGMV and TMV caused green mosaic in cv. Samsun. PMMoV and PaMMV did not infect systemically this tobacco cultivar however the viruses could reproduce in the inoculated leaves, despite the lack of symptoms. Our earlier data revealed that a strain designated as RP101 was systemically spread in some plant of cv. Samsun 60 days after inoculation with P101. RP101 is from pathotype P0 and causes symptomless infection in tomato and tobacco cv. Samsun. It is most probably a mutant of P101 (6). NLL were only developed on the Samsun N N inoculated leaves with ToMV, TMGMV, PaMMV and PMMoV. Strain P101 was also propagated only in the inoculated leaves however the symptoms were temperature dependent. At temperature below 32 ºC there were not symptoms, while NLL appeared at higher temperature. In that case local lesions were much smaller than those resulting from TMV infection. TMV and ToMV caused green mosaic in tomato, P101 - systemic symptomless infection, while TMGMV, PMMoV and PaMMV did not infect tomato at all. However, considerable amount (4.6 % %) of pathotype P0 virions was always detectable in P101 preparations from tomato plants, though in the initial inoculum they were absent (6, 9). Identical data were received after the inoculation of sensitive pepper plants with P101. When P101 was propagated in the three plant species without resistance genes to tobamoviruses, pathotype P0 virions appeared with high frequency. The latter did Biotechnol. & Biotechnol. Eq. 19/2005/2 34 Virology, Supplement

6 not help P101 systemic multiplication in tobacco cv. Samsun, while this was proved in tomato. According Ruiz del Pino et al. the P101 systemic infection of tomato was restricted only in stem tissues and TMV could complement the P101 spreading also in the tomato leaves (10). The presence of pathotype P0 virions in the pepper with no restricted systemic propagation of P101 proposed that the strain possessed high frequency reversed mutations. The presented study of biological properties show that the Bulgarian strain P101 and strain P11 of PaMMV belong to pathotype P1 but differ in their reaction in tomato, tobacco cv. Samsun N N and some C. chinense accessions. The nucleotide sequences of the viral coat protein (CP) gene and 3 noncoding region 3 UTR of P101 are very close to these of PaMMV, sharing values of 98 % and 97 % sequence identity in respective regions compared. On the nucleotide level there are 6 nucleotide changes in each CP ORF and 3 UTR. In the CP ORF four of the substitutions are silent at protein level and the other two lead to amino acid changes a semiconservative Ala-Val at position 145, and a conservative Val-Ile at position 150. The six nucleotide substitutions in the 3 -UTR region lead to the structural differences between both strains (10). These results could explain the electrophoretic and serological differences between P101 and P11. Furthermore, the distinction in the biological properties of both strains probably is due to differences in the nucleotide sequence of other genes. Summarizing the presented data it could be seen, that compared to other tobamoviruses, P101 is the closest to strain P11 of PaMMV. The established differences between the strains, although significant, are not enough to propose P101 as a separate virus. In conclusion, the Bulgarian pepper strain P101 can be related to PaMMV. Acknowledgements The authors thank to Dr. A. T. B. Rast and Dr. F. Garcia-Arenal for the strains granted - P11, U1, U2, GP2 and GP3, and also to the Fund for Scientific Investigations of the Ministry of Education and Science (A- 901/99) for the financial support. REFERENCES 1. C.M.I./A.A.B. Descriptions of plant viruses (1975) 151; (1976) 156, (1988) 330; (1989) Daskalov S., Poulos J.M. (1994) Capsicum and Eggplant Newsletter, 13, Garcia-Luque I., Serra M.T., Alonso E., Wicke B., Ferrari M.L., Diaz-Ruiz J.R. (1990) J. Phytopath., 129, Gilardi P., Wicke B., de la Cruz A., Sarra M.T., Garcia-Luque I. (1999) Recent Res. Devel. Virol., 1, Gooding G.V., Hebert T.T. (1967) Phytopathology, 57, Ivanova E.S. (1982) Doct. Thesis, Moskow (in Russian). 7. Kostova D.P. (1983) Doct. Thesis, Plovdiv (in Bulgarian). 8. Rasr A.Th.B. (1977) In: EUCARPIA Capsicum 77, Avignon-Montfavet, Rast A.Th.B. (1988) Capsicum Newsletter, 7, Ruiz del Pino M., Moreno A., Garcia de Lacoba M., Castillo-Liuva S., Gillardi P., Serra M.T., Garcia-Luque I. (2003) Arch Virol., 148, Stoimenova E. (1983) In: Capsicum and eggplant 83, Plovdiv, Stoimenova E. (1995) J. Culture collections (Sofia), 1, Stoimenova E., Yordanova A., Brumbarov K. (1999) Compt. Rend. Acad. Bulg. Sci., 52 (9-10), Tobias I., Rast A.Th.B., Maa D.Z. (1982) Neth. J. Plant Pathol., 88, Wallenberg B., Andersson U.-B. (1978) Immunoelectrophoretic Techniques with LKB 2117 Multiphor. LKB Application Note 249, LKB-Producter ABB, Sweden. Virology, Supplement 35 Biotechnol. & Biotechnol. Eq. 19/2005/2