AGROINOCULATION METHODS TO SCREEN WILD LYCOPERSICON FOR RESISTANCE TO TOMATO YELLOW LEAF CURL VIRUS

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1 Journal of Plant Pathology (2001), 83 (3), Edizioni ETS Pisa, AGROINOCULATION METHODS TO SCREEN WILD LYCOPERSICON FOR RESISTANCE TO TOMATO YELLOW LEAF CURL VIRUS B. Picó, M. Ferriol, M.J. Díez and F.N. Viñals Department of Biotechnology (Genetics), Universidad Politécnica de Valencia, Camino de Vera 14, 46022, Valencia, Spain SUMMARY The effectiveness of agroinoculation techniques for causing systemic infection by TYLCV in different wild and cultivated Lycopersicon was determined. Rubagroinoculation of leaves, increased the effectiveness of sap-transmission reported previously, but resulted in an erratic and mild infection that did not discriminate among genotypes with different resistance levels. Stem agroinoculation was more effective, and 100% was obtained in the susceptible control. It can be used in breeding programmes as complementary to inoculation using Bemisia tabaci. Both techniques of inoculation provide a precise characterization of the resistance mechanisms in each genotype. Partial resistance to the virus along with resistance to the vector were found in L. hirsutum LA 1777 and L. pimpinellifolium hirsute INRA. The highest levels of virus resistance were observed in three L. chilense accessions (LA 1969, LA 1938, LA 1932). Resistance derived from LA 1932 remained after its introgression into cultivated tomato, giving breeding lines that were highly resistant to TYL- CV. Key words: Begomovirus, TYLCV, Agrobacterium tumefaciens, Lycopersicon chilense, genetic resistance. INTRODUCTION Tomato yellow leaf curl virus (TYLCV; genus Begomovirus, family Geminiviridae) causes up to 100% losses in crop production in many countries around the world (Picó et al., 1996; Czosnek and Laterrot, 1997). Begomoviruses are transmitted by the whitefly Bemisia tabaci (Homoptera: Aleyrodidae). The lack of mechanical transmission prevents adequate screening for resistant sources and renders the subsequent selection process more difficult. Artificial Corresponding author: F.N. Viñals Fax: fnuez@btc.upv.es whitefly-mediated inoculation routines that avoid escapes have been reported (Picó et al., 1998; Vidavsky and Czosnek 1998), but it is difficult to apply uniform inoculum pressure. Variability in conditions sometimes leads to contradictory results, attributing different resistance levels to the same genetic source (Picó et al., 1996 and 1998; Vidavsky et al., 1998). Resistance to the vector, reported in wild Lycopersicon, can mask the existence of virus resistance (Muniyappa et al., 1991; Chanarayappa et al., 1992). Difficulties derived from vector management have encouraged the development of alternative inoculation procedures. Graft-inoculation is effective but tedious for routine s (Friedmann et al., 1998). Agroinoculation uses Agrobacterium tumefaciens to deliver cloned viral DNA into host cells; circular, monomeric viral DNA forms are generated from tandem repeats and spread systemically throughout the plant (Grimsley et al., 1986). Agroinoculation has been successful to introduce geminiviruses into host leaf disks, germinating seeds, and whole plants (Czosnek et al., 1993; Kheyr-Pour et al., 1994), even in species previously considered recalcitrant to Agrobacterium infection, such as cereals (Grimsley et al., 1987). Leaf disk agroinoculation is routinely employed as a simple and fast method to study viral functions and resistance mechanisms, mainly using model species such as Nicotiana spp. However, to use this technique in the screening of tomato and wild Lycopersicon, further studies on transformation and regeneration procedures are required (Czosnek et al., 1993). Agroinoculation of whole plants overcomes these disadvantages. The usual methods employed are based on direct injection of transformed bacterial cultures into the plant vascular system. However, the use of plant agroinoculation in breeding programs has been questioned, because it sidesteps the initial virus-vector-plant interaction and may overlook useful sources of TYLCV resistance (Kheyr-Pour et al., 1994). Genes conferring complete resistance to TYLCV have not been reported to date (Laterrot, 1995; Vidavsky et al., 1998; Picó et al., 1999 b, c). The effectiveness of agroinoculation procedures to differentiate

2 216 Use of agroinoculation to identify TYLCV resistance Journal of Plant Pathology (2001), 83 (3), among different levels of partial resistance therefore needs to be addressed. In the present study, wild and wild-derived Lycopersicon genotypes have been screened by using agroinoculation methods. The response of the different genotypes is compared with their behavior after whitefly inoculation. MATERIALS AND METHODS Plants. The screening was performed using two tomato lines, the susceptible control FC [from the Genebank of the Polytechnic University of Valencia (UPV)], and the L. chilense-derived line BC 1932 (2 backcrossing generations to tomato and 3 selfing generations), and 5 wild Lycopersicon accessions previously reported as partially resistant to TYLCV upon whitefly inoculation (Kasrawi, 1989; Zakay et al., 1991; Picó et al., 1998, 1999c): L. pimpinellifolium hirsute INRA (supplied by Dr. H. Laterrot, INRA, France), and L. hirsutum LA 1777, L. chilense LA 1932, LA1938, and LA 1969 (supplied by Dr. C.M. Rick, Tomato Genetics Resource Center, USA). Agrobacterium culture. A. tumefaciens LBA 4404 bearing a tandem repeat of the TYLCV-Alm (Almeria, Spain) was used in all s. TYLCV-Alm is an isolate of the TYLCV-Sr (Sardinia) species (Navas-Castillo et al., 1999), and was provided by Dr. E.R. Bejarano, University of Malaga, as plasmid pga482. For routine inoculation, bacterial cultures were grown for 48 h at 28ºC in YEB medium supplemented with 100 µg ml -1 rifampicin and 5 µg ml -1 tetracycline. Cells were concentrated tenfold by centrifugation, and immediately used for inoculation. Screening Lycopersicon spp. by agroinoculation. Two experiments were conducted. In the first, carried out over two consecutive years, twenty plants per genotype at the 6 true leaf stage were inoculated using two methods: (i) Leaf agroinoculation (LA), i.e. rub inoculation of the third youngest leaf from the apex, dusted with carborundum, with the A. tumefaciens suspension (Grimsley et al., 1986); (ii) Stem agroinoculation (SA), i.e. injection of the bacterial suspension into the axillary buds of the three youngest leaves (Kheyr-Pour et al., 1994). Measures to prevent accidental release of Agrobacterium into the environment were taken (Elmer et al., 1988). Systemic infection after LA and SA was recorded by sampling apex and stem tissues at 30 days post-inoculation (DPI). Additionally, in a second experiment carried out only in the second year using stem agroinoculation, samples of apex, root and eight sections of the stem were taken at 5, 15 and 30 DPI, to monitor virus spread throughout the plant (six plants were analysed on each sampling date). Symptom scoring. Symptoms were noted weekly. Severity was scored on a scale of 0 (symptomless) to 4 (symptoms as severe as the susceptible control, including leaf yellowing, curling and severe stunting of the plant) as described in Picó et al. (1998). TYLCV detection. The squash-blot procedure was used to detect TYLCV. Sampling, probe radiolabelling, and hybridisation conditions were performed essentially as reported in Picó et al. (1999b). Sections (0.5 cm 2 ) of different plant tissues were directly squashed onto a nylon membrane. Membranes were hybridised with a radiolabelled full-length TYLCV DNA probe, washed once for 10 min in 2 x SSC and 2% SDS at room temperature, and twice for 15 min in 0.1 x SSC and 0.1% SDS at 60ºC. After hybridisation, membranes were exposed to a phosphor imager screen and analysed by densitometry (Bio-imaging analyser, BAS-1500, Fuji film, Tokyo). The infection percentage (number of positive plants by squash blotting over the total number of inoculated plants) was recorded for each genotype. Viral DNA accumulation was quantified relatively to the maximum accumulation in the susceptible control (scale of 0-10). Squash-blotting does not provide accurate quantification of viral DNA, but is useful in differentiating relative viral accumulation among materials with different levels of resistance (Picó et al., 1999b). RESULTS A low infection rate (< 50%) was achieved using the LA method in the susceptible tomato FC (Table 1). Erratic infections were also observed in wild and wild-derived Lycopersicon spp. Necrotic areas appeared on the inoculated leaves of L. pimpinellifolium at 1 DPI. Tissue susceptibility to the bacterial suspension may be the cause of the zero infection found in this genotype. In both s, SA was much more efficient, with > 90% infection in FC and the two wild L. hirsutum LA 1777 and L. pimpinellifolium hirsute INRA (Table 1). Infection percentages in all L. chilense accessions and the line BC 1932, derived from LA 1932, ranged from 40 to 70%. TYLCV was detected in stem sections of all genotypes after 5 DPI (Fig. 1A). Differences in viral accumulation started to appear at 5 DPI and were clear at 15 DPI, when the virus reached the apex and roots of tomato lines FC and BC TYLCV DNA transloca-

3 Journal of Plant Pathology (2001), 83 (3), Picó et al. 217 tion was delayed in wild accessions (Fig. 1B). Maximum TYLCV accumulation at 30 DPI occurred in L. esculentum FC and L. hirsutum LA 1777 (Fig. 1C). Viral concentration in L. pimpinellifolium hirsute INRA was lower by the end of the. Both wild genotypes developed only mild disease compared to the severe symptoms found in the susceptible control (Table 1). TYLCV DNA accumulation in L. chilense accessions and in the L. chilense-derived line BC 1932 at 30 DPI was significantly lower than in the susceptible control and the other wild accessions (Fig. 1C). The pattern of virus accumulation in BC 1932 was similar to that of the susceptible control, but with extremely low viral accumulation. Plants of this accession remained symptomless or with mild symptoms during both s. Table 1. TYLCV infection percentages after agroinoculation of Lycopersicon spp. showing variable levels of resistance. Genotype a Percentage of plants squash-blot positive at 30 days post-inoculation (DPI). 20 plants were used per genotype for each. Numbers in brackets indicate the average symptom rating at 30 DPI, scored from 0 (no symptoms) to 4 (severe symptoms). DISCUSSION Agroinoculation Leaf First Second Stem First Second L. pimpinellifolium 00 a (0.0) 00 (0.0) 090 (1.5) 100 (2.0) hirsute INRA L. hirsutum LA (0.0) 20 (0.0) 100 (1.0) 100 (1.5) L. chilense LA 1938 LA 1932 LA (0.0) 05 (0.0) 19 (0.0) 10 (0.0) 10 (0.0) 05 (0.0) 044 (0.5) 050 (0.5) 056 (0.5) 070 (0.0) 060 (0.0) 040 (0.5) L. esculentum BC (0.5) 40 (0.5) 070 (1.0) 060 (1.0) L. esculentum FC 45 (2.5) 50 (3.0) 095 (4.0) 100 (4.0) The agroinoculation procedures LA and SA differed in their capacity to induce systemic infection in tomato and wild Lycopersicon partially resistant to TYLCV. TYLCV can be mechanically transmitted with low efficiency (< 8%) to tomato plants using sap from infected plants (Makkouk et al., 1979). The use of Agrobacterium as a vector to transfer TYLCV increased the effectiveness of sap-inoculation in our s. However, the poor efficiency of LA inoculation resulted in mild infections that did not differentiate among genotypes with different levels of resistance. The damage caused by bacterial inoculum to leaf tissues of L. pimpinellifolium hirsute INRA is consistent with the susceptibility of this genotype to bacterial transformation observed in previous leaf disk agroinoculation s (Picó et al., 1999a). In SA, the virus is introduced directly into vascular tissue from which long-distance transport via sieve elements occurs, particularly to the shoot apex, young tissues, and roots. This movement has been well characterized for many viruses and is reportedly very rapid (Carrington et al., 1996). Indeed, the large amounts of TYLCV injected into the stem moved rapidly from the point of inoculation. TYLCV DNA was detected in stem sections as soon as 2 DPI, earlier than after whitefly-mediated inoculation (Michelson et al., 1994; Rom et al., 1993). The large amounts of viral DNA found in L. hirsutum LA 1777 suggest that the immunity to TYLCV reported in this accession upon whitefly inoculation (Zakay et al., 1991) was probably due to resistance to the vector. Both mechanisms, antixenosis and antibiosis, have been reported in L. hirsutum (Muniyappa et al., 1991; Channarayappa et al., 1992). Despite the high viral accumulation, LA 1777 remained symptomless, hence, a high level of tolerance to TYLCV accompanies vector resistance mechanisms. This behaviour agrees with data from leaf-disk agroinoculation (Picó et al., 1999a) and graft inoculation with TYLCV-Sr (Fargette et al., 1996). High levels of viral accumulation have also been reported after agroinoculation of LA 1777 with TYLCV-Is isolates (Kheyr-Pour et al., 1994). However, in this study the authors found a lower level of tolerance, as infected plants developed mild symptoms. The differential response against both isolates should be taken into account in breeding programs for TYLCV resistance conducted in areas, such as Spain, where mixed infections with TYLCV-Sr and TYLCV-Is are frequent (Navas-Castillo et al., 1999). SA allowed screening of genotypes such as L. pimpinellifolium hirsute INRA that responded poorly to LA and leaf disk agroinoculation (Picó et al., 1999a). Virus accumulation after SA was similar to that found after graft inoculation with TYLCV-Is and Sr (Kasrawi, 1989; Fargette et al., 1996). The reduced rate of virus accumulation compared to the susceptible control confirms the presence of resistance mechanisms to the virus. This accession has been also reported as partially resistant to the vector (Kasrawi and Mansour, 1994). Therefore, its response to whitefly inoculation is due to resistance to both the vector and the virus (Zakay et al., 1991). L. chilense accessions showed the highest levels of resistance to TYLCV-Sr. After SA, TYLCV was detected in inoculation sites but not in distal tissues. The virus was confined to inoculation areas throughout the

4 218 Use of agroinoculation to identify TYLCV resistance Journal of Plant Pathology (2001), 83 (3), Fig. 1. TYLCV DNA accumulation in different plant tissues of several Lycopersicon spp. after stem agroinoculation (A: 5 days post-inoculation; B: 15 days post-inoculation; C: 30 days post-inoculation). Virus accumulation was scored relative to the maximum accumulation in the susceptible control at 30 DPI (10). Values represent the average for 6 plants; bars indicate the standard error. Only the results of L. chilense accession LA 1932 have been included as the others (LA 1938 and LA 1969), behaved similarly.

5 Journal of Plant Pathology (2001), 83 (3), Picó et al day experiment. These results differ from those of Kheyr-Pour et al. (1994), who reported significant viral accumulation after stem agroinoculation of LA Discrepancies may be due to a higher virulence of TYLCV-Is isolates (Navas-Castillo et al., 1999). Our results are also supported by other experiments, in which very low levels of viral DNA have been detected in these and other L. chilense accessions inoculated by Bemisa tabaci (Picó et al., 1999c). Resistance derived from accession LA 1969 has already been characterized. It is controlled by a single partially dominant gene, Ty-1, that causes the inhibition of both virus replication and long-distance movement upon whitefly inoculation (Michelson et al., 1994). The genetics of the resistance derived from LA 1932 and LA 1938 has not been precisely elucidated. Griffiths (1998) found differences between LA 1932 and LA 1969 in resistance to a closely related Begomovirus, Tomato mottle virus (ToMoV). This author also found molecular markers linked to ToMoV and TYLCV resistance, which were polymorphic between 1932 and Although the Ty-1 gene is also present in LA 1932 different modifier genes could be altering the response of each accession to TYLCV. Regarding resistance mechanisms, previous s with agroinoculated leaf-disks showed that tissues of both L. chilense 1932 and LA 1969 permit similar reduced virus accumulation compared to susceptible tomato genotypes (Picó et al., 1999a). The high level of resistance found in these L. chilense accessions remained after introgression in L. esculentum. The behavior of BC 1932 was more similar to that of the susceptible control, in which TYLCV moved from inoculated leaves to the apex and roots. However, in FC, virus concentration increased from 15 DPI to 30 DPI, whereas it decreased in BC Both reduced viral replication and/or inhibition of short and long distance translocation may account for this different behavior. This advanced breeding line, BC 1932, which is readily crossed with the cultivated tomato, has been selected as a resistant source to introgress TYL- CV resistance in commercially interesting tomato cultivars. ACKNOWLEDGMENTS The authors thank Dr E.R. Bejarano (University of Malaga, Spain) for kindly providing the transformed bacteria for agroinoculation and the TYLCV-Sr probe for hybridisation. This work was supported by project CICYT no. AGF C REFERENCES Carrington J.C., Kasschau K.D., Mahajan S.K., Schaad M.C., Cell to cell and long distance transport of viruses in plants. The Plant Cell 8: Chanarayappa C., Shivashankar G., Muniyappa V., Frist RH., Resistance of Lycopersicon species to Bemisia tabaci, a tomato leaf curl virus vector. Canadian Journal of Botany 70: Czosnek H., Kheyr-Pour A., Gronenborn B., Remetz E., Zeidan M., Altman A., Rabinowitch H.D., Vidavsky S., Kedar N., Gafni Y., Zamir D., Replication of Tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes. Plant Molecular Biology 22: Czosnek H., Laterrot H., A worldwide survey of tomato yellow leaf curl viruses. Archives of Virology 142: Elmer J.S., Sunter G., Gardiner W.E., Brand L., Browning C.K., Bisaro D.M., Rogers S.G., Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs. Plant Molecular Biology 10: Fargette D., Leslie M., Harrison B.D., Serological studies on the accumulation and localization of three tomato leaf curl geminiviruses in resistant and susceptible Lycopersicon species and tomato cultivars. Annals of Applied Biology 128: Friedmann M., Lapidot M., Cohen S., Pilowsky M., Novel source of resistance to Tomato yellow leaf curl virus exhibiting a symptomless reaction to viral infecction. Journal of the American Society for Horticultural Science 123: Griffiths P.H., Inheritance and linkage of geminivirus resistance genes derived from Lycopersicon chilense (Dunal) in tomato (Lycopersicon exculentum Mill.). Ph. D. Thesis. University of Florida, USA. Grimsley N., Hohn T., Davies J.W., Hohn B., Agrobacterium-mediated delivery of infectious maize streak virus into maize plants. Nature 325: Grimsley N., Hohn B., Hohn T., Walden R., Agroinfection, an alternative route for viral infection of plants by using the Ti plasmids. Proceedings of the National Academy for Horticultural Science 83: Kasrawi M.A., Inheritance of resistance to Tomato yellow leaf curl virus (TYLCV) in Lycopersicon pimpinellifolium. Plant Disease 73: Kasrawi M.A., Mansour A., Genetics of resistance to Tomato yellow leaf curl virus in tomato. Journal of Horticultural Science 69: Kheyr-Pour A., Gronenborn B., Czosneck S., Agroinoculation of Tomato yellow leaf curl virus (TYLCV) overcomes the virus resistance of wild Lycopersicon species. Plant Breeding 112:

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