Euphytica (2011) 180:307–319 DOI 10.1007/s10681-011-0355-8
Search in Solanum (section Lycopersicon) germplasm for sources of broad-spectrum resistance to four Tospovirus species E´. C. Dianese • M. E. N. Fonseca • A. K. Inoue-Nagata • R. O. Resende L. S. Boiteux
•
Received: 23 September 2010 / Accepted: 17 January 2011 / Published online: 29 January 2011 Ó Springer Science+Business Media B.V. 2011
Abstract The genus Tospovirus was considered as monotypic with Tomato spotted wilt virus (TSWV) being the only assigned species. However, extensive studies with worldwide isolates revealed that this genus comprises a number of species with distinct virulence profiles. The Neotropical South America is one center of Tospovirus diversity with many endemic species. Groundnut ringspot virus (GRSV), TSWV, Tomato chlorotic spot virus (TCSV), and Chrysanthemum stem necrosis virus (CSNV) are the predominant tomato-infecting species in Brazil. Sources of resistance were found in Solanum (section Lycopersicon) mainly against TSWV isolates from distinct continents, but there is an overall lack of information about resistance to other viral species. One-hundred and five Solanum (section Lycopersicon: Solanaceae) accessions were initially evaluated for their reaction against a GRSV isolate by analysis of symptom expression and systemic virus accumulation using DAS-ELISA. A subgroup comprising the most resistant accessions was re-evaluated in a second assay with TSWV, TCSV, and GRSV isolates E´. C. Dianese R. O. Resende (&) Departamento de Fitopatologia, Universidade de Brası´lia (UnB), 70910-900, Brası´lia, DF, Brazil e-mail:
[email protected] M. E. N. Fonseca A. K. Inoue-Nagata L. S. Boiteux National Center for Vegetable Crops Research (CNPH), Empresa Brasileira de Pesquisa Agropecua´ria (Embrapa Hortalic¸as), CP 218, 70359-970 Brası´lia, DF, Brazil
and in a third assay with a CSNV isolate. Seven S. peruvianum accessions displayed a broad-spectrum resistance to all viral species with all plants being free of symptoms and systemic infection. Sources of resistance were also found in tomato cultivars with the Sw-5 gene and also in accessions of S. pimpinellifolium, S. chilense, S. arcanum, S. habrochaites, S. corneliomuelleri, and S. lycopersicum. The introgression/incorporation of these genetic factors into cultivated tomato varieties might allow the development of genetic materials with broad-spectrum resistance, as well as with improved levels of phenotypic expression. Keywords Tospovirus Resistance Germplasm Wild species Tomato
Introduction A number of thrips-transmitted Tospovirus species (family Bunyaviridae) is associated with economically important diseases of fresh-market and processing tomatoes (Solanum lycopersicum L.) around the world (Silberschmidt 1937; Prins and Goldbach 1998; Williams et al. 2001; Jones 2005; Pappu et al. 2009; Giordano et al. 2010). The genus Tospovirus was initially described as monotypic, with Tomato spotted wilt virus (TSWV) being ´ vila et al. 1993). assigned as the sole species (de A
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However, extensive biological and molecular characterization of worldwide isolates revealed that this ´ vila genus comprises a complex of viral species (de A et al. 1993; Pozzer et al. 1996; Bezerra et al. 1999; Whitfield et al. 2005). Currently, besides the typespecies Tomato spotted wilt virus, there are at least a dozen of other recognized species in this genus (Hassani-Mehraban et al. 2005; Jones 2005; Lin et al. 2005; Whitfield et al. 2005; Ciuffo et al. 2008; Dong et al. 2008; Ciuffo et al. 2009; Hassani-Mehraban et al. 2010). The genetic diversity of Tospovirus species in Brazil is one of the highest, suggesting that South America is likely to be one of the centers of origin of this genus (Bezerra et al. 1999). Extensive surveys indicated that Groundnut ringspot virus (GRSV), TSWV, Tomato chlorotic spot virus (TCSV), and Chrysanthemum stem necrosis virus (CSNV) are the prevalent tomato-infecting tospoviruses in tropical and sub-tropical areas of South America (Pozzer et al. 1996; Nagata et al. 1998; Williams et al. 2001; Giordano et al. 2010). In Brazil, infection by Tospovirus species has been a continuous problem since the establishment of commercial fresh-market tomato cultivation in the beginning of the last century (Costa and Kiehl 1938; Costa and Forster 1941; Melo et al. 2009). Yield losses in susceptible tomato cultivars might be as high as 95% under favorable environmental conditions such as those found in the near-equatorial Northeast region of Brazil (Giordano et al. 2000). Cultural and chemical control strategies are limited due to several reasons, including the large number of alternative host plants serving as virus reservoirs, year-round presence of viruliferous thripsvectors, inefficient insecticide and biological control of these insects, and continuous tomato cropping. In this scenario, the use of cultivars with genetic resistance is one of the main strategies employed for disease management in tomatoes (Soler et al. 2003; Gordillo et al. 2008). Over the years, several sources of genetic resistance have been found in accessions of Solanum (section Lycopersicon: Solanaceae) evaluated mainly against TSWV isolates from distinct geographical locations (Smith 1944; Kikuta and Frazier 1946; Homes 1948; Nagai 1975; Cupertino et al. 1986; Paterson et al. 1989; Maluf et al. 1991; Iizuka et al. 1993; Nagai 1993; Stevens et al. 1994; Canady et al. 2001; Lourenc¸a˜o et al. 1997; 2005; Gordillo
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et al. 2008). Saidi and Warade (2008) provide a comprehensive list of the sources of resistance identified in accessions of distinct Solanum (section Lycopersicon) species. The genetic nature of resistance to TSWV isolates in S. lycopersicum was initially found to be controlled by five loci identified as Sw-1a, Sw-1b, sw-2, sw-3, and sw-4 (Finlay 1952, 1953). After that, an additional locus (named Sw-5) was introgressed from S. peruvianum into the South African cultivar ‘Stevens’ (Van Zijl et al. 1986; Stevens et al. 1992). The Sw-5 gene controls a broad-spectrum resistance to a number of TSWV, TCSV, and GRSV isolates (Stevens et al. 1992; Boiteux and Giordano 1993; Brommonschenkel et al. 2000). Additional TSWV resistance genes were found in S. peruvianum ‘UPV 1’ (named as Sw-6) and in S. chilense ‘LA 1938’ (named as Sw-7) and they were introgressed into cultivated tomato (Rosello´ et al. 2001; Canady et al. 2001; Stevens et al. 2007). However, no information is available so far about the resistance spectrum of the Sw-6 and Sw-7 genes. The dominant nature of the Sw-5 gene allowed its massive use in the development of hybrid cultivars. It was found that cultivars carrying the Sw-5 locus had broad-spectrum resistance to distinct TSWV isolates (Stevens et al. 1992; Rosello´ et al. 1998) and also to the related species of the former serogroup II— GRSV and TCSV (Boiteux and Giordano 1993; Boiteux et al. 1993). In fact, the use of commercial tomato cultivars with the Sw-5 gene has had significant positive impact in some areas, avoiding drastic reduction in fruit yield and severe economic losses under high viruliferous thrips pressure (e.g. Giordano et al. 2000). However, it was observed that the Sw-5 gene does not have a complete penetrance (Stevens et al. 1992), allowing the occurrence of ring spot symptoms in the fruits and/or severe systemic necrosis affecting the entire plant after a latent postinoculation period (Stevens et al. 1992; Boiteux and Giordano 1993; Lourenc¸a˜o et al. 1997). The phenotypic expression of the Sw-5-mediated resistance might also show certain levels of phenotypic instability when it is exposed to extreme temperature variation during the day-time, an environmental condition commonly observed in Southeast and Central Brazil (Lourenc¸a˜o et al. 1997). Other important observation is the worldwide occurrence of new TSWV isolates and/or other species able to break
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down the resistance controlled by the Sw-5 gene (Cho et al. 1996; Lathan and Jones 1998; McMichael et al. 2002; Aramburu and Marti 2003; Ciuffo et al. 2005; Bubici et al. 2008). The emergence of isolates virulent to the Sw-5 gene, as well as the new set of viral species able to infect tomatoes, demands a pre-emptive search for alternative sources of resistance. Therefore, the objective of this work was to evaluate the reaction of plants from a collection of wild and cultivated tomato accessions to four Tospovirus species endemic to the Neotropical areas of South America. The major aim of the present work was to detect new resistance sources with broad-spectrum resistance to distinct Tospovirus species as well as to find materials with improved levels of phenotypic expression that would be suitable for tomato breeding programs.
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classified according to the current taxonomic rules proposed by Peralta et al. (2005). Accessions corresponding to lines derived from interspecific crossings were also evaluated. The accessions were sown on 128 cells styrofoam trays containing a substrate and maintained in a greenhouse. After 20–30 days, when the seedlings presented at least a pair of true leaves, they were transplanted to 3 l pots containing steamed soil. For the first experiment, three plants per pot and three pots per accession were prepared for each test. One pot was selected as the negative control. All accessions were dusted with carborundum (400 mesh) and all leaves mechanically inoculated with the preparation previously made with the infected leaves used as inoculum source. Following this procedure, the leaf tissue was gently washed to remove the inoculation buffer. After 2 days a second inoculation was done in order to minimize the occurrence of escapes.
Materials and methods Tospovirus isolates The isolates used in this experiment were selected from samples previously stored in a deep freezer (–80°C) and confirmed through ELISA. Four isolates were used on the tests: TCSV (‘BR-03’), GRSV (‘SA-05’), CSNV (‘Chr-1), and TSWV (‘BR-01’). These isolates were mechanically inoculated on Datura stramonium L. and Nicotiana benthamiana Domin. 10–15 days prior to use on the test material. Plants showing heavy symptoms were used as extract on inoculation buffer composed of 0.01 M sodium phosphate (NaH2PO4H20) and 1% sodium sulfite (Na2SO3) (w/v) and distilled water (pH 7.0) for mechanical inoculation of the accessions. The plant tissue/buffer ratio was 1:10. Plant material and screening assays All experiments were conducted at CNPH greenhouses in Brası´lia-DF, with day-time temperature variation between 20 and 40°C. Initially, all accessions were tested with the GRSV isolate, which is the predominant Tospovirus species affecting tomato production in South America (Williams et al. 2001). This first assay (Table 1) was composed of 105 accessions of nine wild and cultivated Solanum (section Lycopersicon) species and botanic varieties
Evaluation of symptom expression and systemic infection after GRSV inoculation All accessions evaluated against the GRSV isolate were analyzed through symptom evaluation based on the following scale: 1 = no symptom or presence of tiny local lesions with no systemic symptoms; 2 = presence of local lesions and mild systemic symptoms; 3 = local lesions and/or severe systemic symptoms. These scores were given based upon visual analysis of all inoculated plants through a period of 30 days after inoculation. The cultivar ‘Viradoro’, carrying the Sw-5 locus in homozygous condition (Giordano et al. 2000), was used as resistant control and ‘Angela Hiper’ (Nagai 1993) was used as susceptible control. Systemic GRSV accumulation was verified 15 days after inoculation through DAS-ELISA (Clark and Adams 1977) using specific anti-sera and the following protocol on 96-wells ELISA plates: 200 ll of 1.0 lg/ml IgG on sodium carbonate coating buffer per well. The plates were then incubated overnight at 4°C. The next steps were adapted from procedures described by Hsu (2009), consisting of three washes with PBS-Tween, 100 ll of extracted samples on PBS-Tween 1:10 proportion, incubation for 2–4 h at 37°C, three washes with PBS-Tween following the addition of 100 ll of 1.0 lg/ml IgG alkaline-phosphatase conjugate on PBS-Tween and incubation at 37°C (2–4 h).
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Table 1 Reaction of Solanum (section Lycopersicon) accessions to an isolate of Groundnut ringspot virus (GRSV) through mechanical inoculation. The subgroup selected for further testing is marked in bold Accession name
Solanum (section Lycopersicon) species
Number of ELISA positive plants/ total inoculated plants
Disease scorea
Angela Hiperb
S. lycopersicum
06/06
3
Viradoro (Sw-5)c
S. lycopersicum
00/06
1
Alambra F1
S. lycopersicum
06/06
3
Rutgers
S. lycopersicum
06/06
3
Duradoro F1 (Sw-5/sw-5)
S. lycopersicum
00/06
1
Tospodoro (Sw-5/Sw-5)
S. lycopersicum
00/06
1
IPA-5
S. lycopersicum
06/06
3
IPA-6
S. lycopersicum
06/06
3
Santa Clara
S. lycopersicum
06/06
3
Santa Clara IL (Sw-5/Sw-5)
S. lycopersicum
00/06
1
Kada Gigante De la Plata
S. lycopersicum S. lycopersicum
06/06 05/05
3 3
Silvestre de Piranga Silvestre de Araxa´
S. lycopersicum var. cerasiforme
02/05
3
S. lycopersicum var. cerasiforme
03/05
3
Silvestre de Felixlaˆndia Silvestre de V. Santo Anta˜o Prı´ncipe Gigante
S. lycopersicum var. cerasiforme
05/05
3
S. lycopersicum var. cerasiforme
05/05
3
S. lycopersicum
06/06
3
PI 306811-67-N-4
S. peruvianum
04/11
2
PI 732293-Nagai #1
S. lycopersicum 9 S. pimpinellifolium
04/05
3
PI 732293-Nagai #2 ˆ ngela Gigante I5100 A
S. lycopersicum 9 S. pimpinellifolium
05/05
3
S. lycopersicum
06/06
3
Silvestre Altamira
S. lycopersicum var. cerasiforme
05/05
3
Silvestre de Vito´ria ˆ ngela Gigante A
S. lycopersicum var. cerasiforme
04/04
3
S. lycopersicum
06/06
3
PI 732293-Nagai #3 Silvestre do Para´ LA1967
S. lycopersicum 9 S. pimpinellifolium
05/05
3
S. lycopersicum var. cerasiforme S. chilense
04/05 00/06
3 1
PI 126445
S. habrochaites
01/01
3
PI 126931
S. pimpinelifolium
06/06
3
PI 127827
S. habrochaites
02/05
2
LA258
S. lycopersicum
00/05
1
Rick 3-71
S. lycopersicum
04/05
3
LA1663
S. lycopersicum
05/05
3
CNPH 0457/RLT
S. lycopersicum
07/11
2
PI 279567 (Pearl Harbor)
S. lycopersicum
05/05
3
IVT-3
S. lycopersicum 9 S. peruvianum
01/05
2
Niva/USSR
S. peruvianum
03/06
3
Quil-Quil
S. lycopersicum
05/05
3
Silvestre de Brası´lia
S. lycopersicum var. cerasiforme
03/05
3
Silvestre de Calambau´ Silvestre de Ipua˜
S. lycopersicum var. cerasiforme
05/05
3
Pearl Harbor
S. lycopersicum var. cerasiforme S. lycopersicum
01/05 05/05
3 3
VC8 Martinus
S. pimpinellifolium
01/05
2
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Table 1 continued Accession name
Solanum (section Lycopersicon) species
Number of ELISA positive plants/ total inoculated plants
Disease scorea
CGO 6708
S. peruvianum
02/11
2
CGO 6707
S. peruvianum
06/06
3
CGO 6712
S. peruvianum
02/05
3
CGO 6713
S. peruvianum
03/11
2
CGO 6714
S. peruvianum
03/11
2
LA1341
S. pimpinellifolium
06/06
3
LA1616
S. peruvianum
06/06
3
LS 121 Japan
S. peruvianum
02/05
3
Stevens (Sw-5)
S. lycopersicum
01/06
1
LA1270
S. peruvianum
01/06
1
LA1333
S. peruvianum
00/05
1
LA1677
S. peruvianum
00/11
1
WYR 2020
S. peruvianum
03/05
3
WYR 3957 LA111
S. peruvianum S. peruvianum
05/05 05/11
3 2
LA385
S. arcanum
00/05
1
LA1113-1
S. corneliomuelleri
04/11
2
LA1113-2
S. corneliomuelleri
04/11
2
LA1113-3
S. corneliomuelleri
02/05
2
ID 8623
S. peruvianum
03/05
3
ID 8624
S. peruvianum
01/05
3
LA1036
S. chmielewskii
05/05
3
LA1716
S. neorickii
05/05
3
TX 410 (SA 3 LA462)
S. lycopersicum 9 S. peruvianum
00/05
1
CNPH 0951/RLT/Japan
S. lycopersicum
03/05
3
Japan/Iizuka
S. pimpinellifolium
02/05
3
PI 732293-Nagai #4
S. lycopersicum 9 S. pimpinellifolium
00/05
1
PI 319369
S. peruvianum
00/05
1
PI 390715
S. peruvianum
01/05
2
PI 390687 PI 118784
S. peruvianum
05/05
3
S. peruvianum
01/05
2
PI 127813
S. peruvianum
03/05
3
CNPH 1034
S. habrochaites
04/06
3
CNPH 1035
S. peruvianum
03/06
3
CNPH 1036
S. peruvianum
02/06
2
[LA1969 9 Lignon] A7
S. lycopersicum 9 S. chilense
05/05
3
[LA1969 9 Lignon] A49
S. lycopersicum 9 S. chilense
05/05
3
[LA1969 9 Lignon] C23
S. lycopersicum 9 S. chilense
04/05
3
CGO 8200
S. peruvianum
02/06
2
CGO 7650
S. pimpinellifolium
06/06
3
PI 128660
S. peruvianum
04/06
3
H 1548
S. lycopersicum
06/06
3
LA107
S. peruvianum
02/04
3
LA153
S. peruvianum
00/05
1
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Table 1 continued Accession name
Solanum (section Lycopersicon) species
Number of ELISA positive plants/ total inoculated plants
Disease scorea
LA444
S. peruvianum
03/05
3
LA1274
S. peruvianum
02/05
3
LA1331
S. peruvianum
02/02
3
LA1339
S. peruvianum
02/05
3
LA1351
S. peruvianum
05/05
3
LA1609
S. peruvianum
00/05
1
LA1954
S. peruvianum
03/05
3
LA1984
S. peruvianum
02/05
3
LA2152
S. arcanum
00/05
1
LA2157
S. peruvianum
02/05
2
LA2163
S. peruvianum
04/05
3
LA2172
S. peruvianum
03/05
3
LA2185
S. peruvianum
05/05
3
LA2328 LA2732
S. peruvianum S. peruvianum
05/05 01/05
3 2
LA2744
S. peruvianum
00/05
1
LA2964
S. peruvianum
00/05
1
BHRS 1,2
S. lycopersicum
03/05
3
PI 203230 (RLT)
S. lycopersicum
00/08
1
a
Grades: 1—no symptoms or tiny local lesions with no systemic spread, 2—presence of local lesions and mild systemic symptoms, and 3—local lesions and severe systemic spread of the symptoms
b
Susceptible control
c
Resistant control (carrying the Sw-5 locus in homozygous condition)
The reaction was visualized after three washes with PBS-Tween and addition of 100 ll per well of the alkaline phosphatase substrate diluted on diethanolamine buffer (1 mg/ml). The plate reading was done using a Multiscan Bichromatic ELISA reader at 405 nm. A plant was considered as susceptible when the absorbance value was at least two times the absorbance reading of the healthy control.
sub-group of accessions displaying low frequency of systemically-infected plants in the second assay was also used for evaluation in a third assay with a CSNV isolate (Table 3). Inoculation and evaluation procedures were conducted essentially as described above for GRSV.
Results Evaluation of GRSV-resistant plants with other Tospovirus species A sub-set of accessions scored for symptom expression with grades 1 and 2, combined with low frequency of GRSV-systemically infected plants, was either selfed or sib-mated and the seeds were then re-tested in a second assay with isolates of other two Tospovirus species (TSWV and TCSV), and also re-evaluated for the GRSV isolate (Table 2). Another
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One hundred and five accessions belonging to nine Solanum (section Lycopersicon) species and botanical varieties were initially tested for resistance only to GRSV, using mechanical inoculation (Table 1). This strategy was used to simplify the screening process, even though it could result in elimination of some potential sources of resistance specific for one of the other three tospoviruses used in the present work. The reason to choose the GRSV isolate for this initial screening was the importance of this viral species,
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Table 2 Reaction against isolates of three Tospovirus species of the susceptible and resistant controls (‘Angela Hiper’ and ‘Viradoro’) and a group of 24 Solanum (section Lycopersicon) accessions previously selected for resistance Accession name
Solanum (section Lycopersicon) species
Number of ELISA positive plants/total number of plants GRSV
TSWV
TCSV
Angela Hipera
S. lycopersicum
19/21
22/22
18/21
Viradoro (Sw-5)
S. lycopersicum
00/06
00/06
00/06
PI 306811-67-N-4
S. peruvianum
01/21
00/21
00/21
LA1967
S. chilense
04/28
03/22
03/27
PI 127827
S. habrochaites
00/28
06/27
01/22
LA258
S. lycopersicum
01/18
00/13
02/12
CGO 6708
S. peruvianum
00/28
00/27
00/25
CGO 6713
S. peruvianum
00/28
00/27
01/27
CGO 6714
S. peruvianum
00/28
00/21
00/21
LA1270
S. peruvianum
00/20
00/20
00/23
LA1333 LA1677
S. peruvianum S. peruvianum
03/22 03/28
01/16 02/22
01/19 01/22
LA111
S. peruvianum
02/25
00/24
01/18
LA385
S. arcanum
05/15
00/03
01/08
LA1113-1
S. corneliomuelleri
00/20
01/22
01/22
LA1113-2
S. corneliomuelleri
01/21
01/26
02/26
TX 410 (SA 9 LA 462)
(S. lycopersicum 9 S. peruvianum)
03/14
00/08
01/08
PI 732292-Nagai #4
(S. lycopersicum 9 S. pimpinellifolium)
01/20
01/14
01/14
PI 319369
S. peruvianum
00/13
00/16
00/16
CNPH 1036
S. peruvianum
00/28
00/27
00/26
CGO 8200
S. peruvianum
00/28
00/22
00/22
LA136
S. peruvianum
00/09
02/16
01/12
LA1937
S. peruvianum
00/17
01/16
00/14
LA2152
S. arcanum
00/13
02/09
01/05
LA2744
S. peruvianum
00/13
00/09
00/04
PI 203230
S. lycopersicum
00/09
00/10
00/08
Accessions were evaluated against Groundnut ringspot virus (GRSV), Tomato spotted wilt virus (TSWV), and Tomato chlorotic spot virus (TCSV) using mechanical inoculation. Systemic infection was verified by ELISA a
Susceptible control
due to its wide geographical distribution and economic damage caused on the tomato crop in South America (Williams et al. 2001). The mechanical inoculation combined with ELISA enabled the identification of new hosts, as well as accessions with plants free of symptoms and with either low frequency of susceptible plants or mild symptoms. The inoculum viability was confirmed by the susceptible response of the controls (D. stramonium, N. benthamiana, and tomato ‘Angela Hiper’). In this test, a large number of accessions showed high susceptibility to GRSV with close to 100% of the
plants expressing severe symptoms and systemic accumulation of the virus (Table 1). Accessions with low frequency of systemicallyinfected plants and symptom score equal or below 2 were classified as resistant to GRSV (Table 1). This classification was done based upon the presence in the accessions of one or more of the following resistant-like phenotypes: (1) the presence of an immunity type response (i.e. complete absence of symptoms and no systemic symptoms); (2) the presence of small/medium necrotic lesions on inoculated leaves with no systemic symptoms (Fig. 1);
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Table 3 Reaction of ‘Angela Hiper’, ‘Viradoro’, and of a group of 14 Solanum (section Lycopersicon) accessions to one Chrysanthemum stem necrosis virus (CSNV) isolate using mechanical inoculation Accession name
Solanum (section Lycopersicon) species
Number of ELISA positive plants/total number of plants
Angela Hipera
S. lycopersicum
12/12
Viradoro (Sw-5)
S. lycopersicum
00/06
PI 306811-67-N-4 S. peruvianum
00/15
LA1967
S. chilense
04/10
PI 127827
S. habrochaites
00/11
CGO 6708
S. peruvianum
00/09
CGO 6713
S. peruvianum
00/11
CGO 6714
S. peruvianum
00/15
LA1270
S. peruvianum
01/11
LA1677
S. peruvianum
00/11
LA111
S. peruvianum
00/08
LA1113-1
S. corneliomuelleri
00/06
LA1113-2 CNPH 1036
S. corneliomuelleri S. peruvianum
00/11 00/11
CGO 8200
S. peruvianum
00/06
PI 203230
S. lycopersicum
00/08
Fig. 1 The Solanum peruvianum accession CNPH 1036 showing local lesions related to resistance reaction after inoculation with the BR-01 isolate of Tomato spotted wilt virus (TSWV). The arrows indicate the lesions caused by the hypersensitive-like reaction
well as to the other two viral species. Similar results were observed for the sub-group of accessions evaluated for resistance to one CSNV isolate in the third assay (Table 3).
Systemic reaction was verified by ELISA a
Susceptible control
Discussion and (3) low frequency of plants with systemic symptoms. These plants were either selfed or sibmated and the seeds were then used in subsequent assays aiming to identify accessions with multiple resistance. A sub-set of these accessions was re-evaluated in the second assay by inoculation and evaluation by visual symptom observation and ELISA using three isolates: TCSV (‘BR-03’), and TSWV (‘BR-01’) and once again with GRSV (‘SA-05’). This second test was done to confirm their resistance to GRSV and verify their resistance spectrum to other Tospovirus species (Table 2). In this second assay, the susceptible controls (D. stramonium, N. benthamiana, and tomato ‘Angela Hiper’) also exhibited severe symptoms, indicating the viability of the inoculum for all three viruses. The great majority of accessions that showed a resistance profile on the first test against GRSV also exhibited a similar response on the second assay, indicating that the accessions are more genetically uniform in relation to resistance factors against this virus as
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The tomato cultivar ‘Viradoro’, which was used as a resistant control in all three tests due to the presence of the Sw-5 gene in homozygous condition (Giordano et al. 2000), displayed a broad-spectrum resistance that was effective against all four viral species (Tables 1, 2, and 3). This is, to our knowledge, the first formal report that lines carrying the Sw-5 gene are also resistant to CSNV. The cultivar ‘Tospodoro’ (Giordano et al. 2010), the hybrid ‘Duradoro’ (heterozygous for the Sw-5 locus) and the line ‘Santa Clara IL’ (Sw-5/Sw-5) confirmed their resistance against GRSV (Table 1). The South African cultivar ‘Stevens’ (the original source of the Sw-5 gene) displayed one infected plant out of six after inoculation with GRSV (Table 1). These observations suggest that the Sw-5 gene is likely to remain useful in tomato breeding programs aiming to develop cultivars with broad-spectrum resistance to Tospovirus species occurring worldwide. It was observed in all assays that many accessions classified as resistant displayed a variable number of
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plants with systemic symptoms and/or systemic virus accumulation after inoculation with distinct Tospovirus species. These results could be explained by either the incomplete penetrance (Stevens et al. 1992) or by the natural genetic variability that occurs within the accessions. This is especially true for accessions of the former S. peruvianum L. complex, a group of species with predominant self-incompatible and cross-pollinated (allogamous) breeding system (Peralta et al. 2005). The great majority of accessions with no signs of infection for all viruses in all assays are classified as belonging to S. peruvianum. This result was somewhat expected, since S. peruvianum is the source of the Sw-5 and Sw-6 genes, which are two of the main resistance sources used on breeding programs worldwide (Stevens et al. 1992; Rosello´ et al. 2001; Soler et al. 2003; Saidi and Warade 2008). Almost all red-fruited accessions of S. pimpinellifolium L., S. lycopersicum var. cerasiforme (a semidomesticated botanic variety) and all cultivars of ‘Santa Cruz’ (S. lycopersicum) variety group (e.g. ‘Santa Clara’, ‘Kada’, ‘Angela Hiper’, ‘Prı´ncipe Gigante’ and other selections of ‘Angela Gigante’) were highly susceptible to GRSV with 100% of the plants exhibiting symptoms. This varietal group was predominant in Brazil from 1940 until 1990, and some of these cultivars are still being used today in many tomato-producing regions of Brazil (Boiteux et al. 2008). The high levels of susceptibility of the ‘Santa Cruz’ group explains the extreme economic importance that the spotted-wilt disease has on the Brazilian tomato agribusiness and all the research effort dedicated in the past decades to incorporate genetic resistance to tospoviruses (Nagai 1993; Boiteux et al. 2008; Melo et al. 2008). The line ‘PI 732292-Nagai #4’ derived from an interspecific cross between S. lycopersicum and S. pimpinellifolium displayed low frequency of plants with systemic infection by GRSV, TCSV and TSWV, but it was not evaluated for CSNV. ‘PI 203230’ and ‘LA258’ were the only accessions of S. lycopersicum that showed no clear symptoms and/or evidence of systemic infection to TSWV as indicated by the negative reaction on ELISA. Somewhat surprising was the reaction of the accession ‘LA258’. It had 100% of plants free of TSWV infection as well as high frequency of resistant plants to GRSV (one infected plant out of 22 plants) and TCSV (2 out of
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12). Additional studies should be conducted with this accession since it has the ‘wooly’ (Wo) mutation, which confers high amount of trichomes in the leaf tissue and stems, that might interfere with the mechanical transmission of the virus. However, it is interesting to point out that the accession ‘LA1663’ has the same Wo mutation, but it was found to be highly susceptible to GRSV (Table 1). The accession ‘PI 203230’, a selection within the variety ‘Rey de Los Tempranos’ (RLT) obtained in Australia, has been already reported as field resistant to Tospovirus isolates (Maluf et al. 1991). On the contrary, two other ‘RLT’-derived accessions (e.g. ‘CNPH 0457’ and ‘CNPH 0951’) displayed a high frequency of plants susceptible to GRSV (Table 1). On the second assay, ‘PI 203230’ had a response classified as resistance to isolates of all three species (Table 2), indicating that the selection previously done was effective to stabilize the resistance factors in this tomato line (Boiteux et al. 2004). It is interesting to note that selections from different origins of the cultivar ‘Pearl Harbor’ (= ‘PI 279567’), which was one of the first sources of TSWV resistance described in the literature (Kikuta and Frazier 1946), were highly susceptible to the GRSV isolate (Table 1), suggesting the presence of isolate- and/or speciesspecific resistance in S. lycopersicum. Solanum chilense (Dunal) Reiche accessions have been already reported as sources of resistance against tospoviruses (Stevens et al. 1994; Canady et al. 2001). Material derived from S. chilense ‘LA1938’ was classified as resistant, even against a Hawaiian TSWV isolate capable of breaking the Sw-5 gene resistance, and advanced lines derived from this accession showed promising results against isolates of this viral species (Canady et al. 2001; Scott et al. 2005). Here, all three lines derived from interspecific crosses with S. chilense ‘LA1969’ were found to be susceptible to GRSV (Table 1).The S. chilense accession ‘LA1967’ displayed a peculiar behavior on the screening assays. On the first evaluation against GRSV, it was classified as resistant. On the second assay, even though not displaying clear symptoms of infection, the result obtained through the ELISA test indicated a high level of virus accumulation in the non-inoculated tissues, indicating a tolerance-like response (sensu Cooper and Jones 1983). However, in relation to CSNV, this accession displayed 40% of symptomatic plants (Table 3).
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Sources of resistance to TSWV have been previously found in S. habrochaites Knapp & Spooner (= Lycopersicon hirsutum Dunal) accessions (Cupertino et al. 1986; Maluf et al. 1991; Kumar et al. 1995). Thrips-resistance was one explanation for the TSWV resistance in S. habrochaites accessions evaluated in the USA (Kumar et al. 1995), and might also be associated with field resistance observed in S. habrochaites ‘PI 127826’ and ‘PI 134417’ in Brazil (Maluf et al. 1991), even though contradictory results have been observed for ‘PI 134417’, which was identified as susceptible in other field evaluation carried out in Brazil (Lourenc¸a˜o et al. 1997). However, as reported here and elsewhere (Cupertino et al. 1986), resistance sources have been found in S. habrochaites after mechanical inoculation, indicating that resistance to the virus may not be only associated with interference with the vector transmission. In our trials, S. habrochaites ‘PI 127827’ was classified as resistant, but its resistance was not classified as broad-spectrum, since the GRSV and TSWV isolates were able to induce systemic symptoms in a variable number of plants of this accession (Tables 1 and 2). Our results confirmed S. peruvianum L. as the most important source of resistance against Tospovirus species that have ample geographic distribution on Neotropical regions of South America. Seven S. peruvianum accessions (‘CGO 6714’, ‘CGO 6708’, ‘CGO 8200’, ‘CNPH 1036’, ‘PI 319369’, ‘LA1270’, and ‘LA2744’) displayed broad-spectrum resistance to all viral species with all plants free of systemic infection. The line ‘TX 410’ derived from an interspecific cross between S. lycopersicum and S. peruvianum displayed also low frequency of plants infected by the three species. The resistance found in this line will allow genetic studies of this resistance employing cultivated tomato as parental lines. To our knowledge the present work is also the first formal report of Tospovirus resistance in S. arcanum Peralta ‘LA2152’ and in S. corneliomuelleri J.F. Macbr ‘LA1113-2’. These accessions belong to two recently described Solanum (section Lycopersicon) species closely related to S. peruvianum (Peralta et al. 2005). Solanum arcanum ‘LA2152’ displayed particularly consistent results against GRSV (no infection in a total of 18 evaluated plants). The study reported here is one of the few involving the evaluation of a large number of Solanum (section Lycopersicon) accessions against a panel of
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Tospovirus species. The most extensive work related to the host variability thus far was conducted by Gordillo et al. (2008), which evaluated 285 accessions of S. peruvianum. However, the pathogen variability was limited to two TSWV isolates (‘TSWV6’ collected in Hawaii and able to infect plants with the Sw-5 gene and ‘Anwa-1’ isolate from Australia, which is avirulent to the Sw-5 gene). Even with these differences, some comparisons could be done between the results obtained by Gordillo et al. (2008) and the present work. In both screenings, the S. peruvianum accessions ‘LA1616’, ‘PI 390687’, and ‘PI 128660’ were found to be susceptible, with a frequency of symptomatic plants ranging from 60 to 100%. These ‘‘universal’’ S. peruvianum susceptible accessions could be useful as parental lines in genetic studies aiming to elucidate the inheritance of resistance to Tospovirus species in many S. peruvianum lines, since interspecific crossings with the cultivated tomato and other susceptible species are difficult. Another important observation is that Gordillo et al. (2008) were not able to identify accessions with 100% of resistant plants. The accession S. peruvianum ‘PI 126946’ was one with the best results displaying 80% of resistant plants. A lower frequency of resistant plants was also observed in S. peruvianum ‘LA1677’ (50%) and S. arcanum ‘LA385’ (none). In contrast, ‘LA385’ and ‘LA1677’ were classified as resistant against all four viral species used in our experiments, even though some symptomatic plants were observed for GRSV in ‘LA385’ (Table 2). Solanum arcanum ‘LA385’ has been also found to be resistant to other three Tospovirus isolates from Brazil (Iizuka et al. 1993). These conflicting results can be explained by distinct virulence profiles of the isolates, direct use by Gordillo et al. (2008) of the original accessions (i.e. without any previous selection) in addition to the fact that the accessions listed in Table 2 have been tested and selected multiple times against different tospovirus isolates since 2003/2004. In fact, the majority of these accessions have already expressed resistant reactions when inoculated with TSWV, TCSV, and CSNV isolates (Boiteux et al. 2004). This previous selection might explain the higher frequency of resistant plants in the accessions identified in the present study, with many of them exhibiting 100% of plants without symptoms and with a negative reaction on ELISA to all four viral species (Tables 2 and 3). For this reason, it is
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likely that other accessions that exhibited variable results on the first assay (i.e. accessions potentially segregating for resistance) could have their resistance gene frequencies increased since they are likely to be not stabilized yet. This is especially true for many of the accessions belonging to species with self-incompatibility and/or other allogamous breeding mechanisms. This continuous selection process would be an effective strategy to allow the gradual increase on the frequency of resistant plants within accessions of S. peruvianum, S. arcanum, S. corneliomuelleri, and S. chilense. The identification of many potential sources in distinct Solanum (section Lycopersicon) species might be useful to diversify the genetic basis and to allow the selection of tomato material with multiple resistance profile to a spectrum of Tospovirus species/ isolates. It is interesting to note that this relatively high number of accessions with broad-spectrum resistance to Tospovirus species in genus Solanum (section Lycopersicon) is in sharp contrast with the situation reported in the genus Capsicum (also Solanaceae) where only sources of TSWV-specific resistance (Tsw gene) have been found so far ´ vila 1994; Boiteux 1995). (Boiteux and de A The dissemination of tospovirus isolates capable of breaking the Sw-5 gene resistance has been reported in different continents (Cho et al. 1996; Lathan and Jones 1998; McMichael et al. 2002; Aramburu and Marti 2003; Ciuffo et al. 2005; Bubici et al. 2008), probably due to the more extensive use of this source on commercial cultivars (Gordillo et al. 2008). This group of Sw-5-resistance breaking isolates is causing major epidemiological concerns, especially due to the severity of this disease to the tomato. In this context, it would be interesting to test the accessions identified here against these isolates. This was not investigated due to the fact that, to our knowledge, these Sw-5-resistance breaking isolates have not been reported in Brazil thus far. If these accessions proved to be resistant also to these ‘‘resistance-breaking’’ isolates, the introduction of their genetic factors into commercial tomatoes would permit their rotation with all effective loci described in tomatoes so far (e.g. Sw-5, Sw-6, and Sw-7), contributing to lower the selection pressure that could establish new tospovirus isolates with a broad virulence profile. It would also be interesting to verify if some of these resistant accessions reported here have similar
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structural features of the Sw-5 locus by comparing their sequences with that of the original source of resistance from S. peruvianum, which was introgressed into the cultivar ‘Stevens’ (Van Zijl et al. 1986; Stevens et al. 1992). Molecular markers specifically targeting the Sw-5 genomic region (Dianese et al. 2010) could be used for this analysis. In addition, the molecular cloning of the genetic factors associated with this broad-spectrum resistance might provide a new set of transgenic strategies to control resistance to other Tospovirus species described so far affecting tomato as well as other vegetable, ornamental, and field crops (Hassani-Mehraban et al. 2005; Whitfield et al. 2005). In summary, alternative sources of broad-spectrum resistance were identified in accessions of S. peruvianum, S. arcanum, S. lycopersicum, S. chilense, and S. corneliomuelleri. The presence of genetic diversity for resistance against tospovirus species is highly desirable for breeding programs. They could be used as alternative genes and ‘piramidized’ in tomato cultivars aiming to avoid damage caused by new isolates and/or species with a broader virulence profile, which may eventually emerge under natural conditions. In addition, the introgression of these genetic factors into the cultivated tomato might allow the development of cultivars with broad-spectrum resistance as well as with improved levels of phenotypic expression. Acknowledgments This work was supported by Embrapa Hortalic¸as and University of Brası´lia (UnB). The first author was supported by CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior) and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). We would like to thank the CNPH technical staff, especially Antonio Francisco Costa, Oneilson Medeiros de Aquino, Lu´cio Fla´vio Barbosa, Hamilton Jose´ Lourenc¸o, and William Pereira Dutra. Maria Esther N. Fonseca, Alice K. Inoue-Nagata, Renato O. Resende, and Leonardo S. Boiteux are CNPq (MCT) fellows.
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