Improvement of Resistance to Late Blight in Hybrid Tomato

Transcription

1 Hort. Environ. Biotechnol. 55(2): DOI /s Research Report ISSN (print) : ISSN (online) : Improvement of Resistance to Late Blight in Hybrid Tomato Duck Hwan Park 1, Yan Zhang 2, and Byung Sup Kim 2* 1 Department of Applied Biology, Kangwon National University, Chuncheon , Korea 2 Department of Plant Science, Gangneung-Wonju National University, Gangneung , Korea *Corresponding author: bskim@gwnu.ac.kr Received April 5, 2013 / Revised January 28, 2014 / Accepted March 10, 2014 C Korean Society for Horticultural Science and Springer 2014 Abstract. Late blight is one of the major diseases in tomato cultivated both in the greenhouse and the field. In this work, we determined the resistance levels of all progeny lines (F 1 to F 3) and a new variety (NV) F 1 to the late blight disease fungus to evaluate these hybrid lines as potential sources of resistance. The experiments were carried out in greenhouse conditions in a completely randomized block design with three replications. Fifteen out of 204 F 2 plants of cross A (AV107-4 L3708) and 13 out of 203 F 2 plants of cross B (Campari 917 L3708) were found to be resistant to late blight in combined data from greenhouse In addition, 5 resistant F 3 lines derived from each of the two crosses A and B were selected for further investigation. By screening the NV F 1 hybrids obtained by pollination of the F 3 lines by 3 male-sterile cultivars (Wonung-1, 2, and 3), two stable resistant hybrids, Wonung-2 A120 and Wonung-3 B23, were finally produced. An examination of the qualitative characters in the NV F 1 hybrids indicated improved fruit weight and quality. Taken together, our screening results indicate that both of these hybrids are good sources of resistance and have high-value horticultural characters, which can be used for tomato breeding programs. Additional key words: breeding program, horticultural characters, male sterile cultivars, resistant hybrids Introduction Late blight, caused by the oomycete Phytophthora infestans (Mont.) debary, is one of the major diseases in tomato and potato (Fry and Goodwin, 1997). This pathogen causes lesions on the aerial plant parts, leading to defoliation and fruit loss, accompanied by the dispersion of airborne asexual sporangia during the growing season (Shattock, 2002). It has severely affected tomato production, particularly at high humidity and cool temperature, which cause outbreaks in tomatoes cultivated in unheated glasshouses (Moreau et al., 1998). In addition, tomato plants are generally regarded as highly susceptible to this disease (Becktell et al., 2005). Thus, this disease has chronically affected the fruit yield and quality of tomato plants. Various protectant fungicides have been commonly used to control late blight in tomato production worldwide. However, chemical control using fungicides has become ineffective against resistant strains, because they were treated too frequently, at 5-12 day intervals (Möller et al., 2009). In addition, fungicides have such adverse effects as environment pollution, and they are not able to control late blight under some conditions that favor heavy disease pressure (Ko, 1994). Because these defects can adversely affect the effectiveness of fungicides, genetic resistance against late blight has been a priority in tomato breeding programs (Zhu et al., 2008). Host resistance is an important element in late blight management due to its long-term economic benefits for small-scale farmers. It also minimizes changes in the population structure of P. infestans, decreasing the likelihood of the development of fungicide resistance (Hakiza, 1999; Mukalazi et al., 2001). Therefore, we tested whether hybrid tomato lines were effective against domestic isolate from the lesions of late blight on foliar in both pot and greenhouse Our data demonstrate that hybrid tomato lines sustained until the 4 th generation had higher disease resistance than the control line. Materials and Methods Plant Materials Plant materials used in this study were comprised of four late blight-resistant tomato genotypes, Campari, AV107-4, L3708, and 917, and hybrids produced among them from cross A (AV107-4 L3708) and cross B (Campari 917

2 Hort. Environ. Biotechnol. 55(2): L3708) at National Institute of Horticultural & Herbal Science (NIHHS). For hybridization, the anthers of male parents were harvested from opened flowers, and the pollen was transferred to the stigmas of female parents by a brush between 9:00 and 11:00 am. Seeds were obtained from fully ripe fruits and used to grow the F 1 progenies. Approximately 200 F 2 progenies were obtained by self-pollination of F 1 plants, followed by production of F 3 progenies. Completely late blight-resistant F 3 lines were crossed with three malesterile cultivars, Wonung-1, 2, and 3 to raise new variety (NV) F 1 progenies. All seeds were sown in plastic trays containing commercial potting mixture (Plant World, Nongwoobio, Suwon, Korea), and maintained under standard growth chamber conditions at 20 C with 60% relative humidity, and 16/8 hours day and night conditions. At the first true leaf stage, seedlings were transplanted into 6 cm pots and placed in a greenhouse at 20 ± 5 C and 50% relative humidity. The F 1 to F 3 and NV F 1 progenies were tested for resistance against late blight under the same conditions. Pathogen Preparation and Inoculation A wild type isolate of P. infestans was obtained from naturally infected tomato leaves in the fields of Gangnung- Wonju University, Gangwon-do, Republic of Korea. The isolate of P. infestans (A2 mating type) was grown on the V8 agar at 20 C for 15 days and then the sporangia were harvested by flooding the plate with sterile distilled water. For zoospore production, sporangial suspensions were kept at 4 C for 1 hour. Final pathogen concentration was adjusted to zoospores per 1 ml and applied to leaf surfaces by a hand sprayer with three replications on the same day. Inoculated plants were covered with plastic vats to increase humidity to ensure successful infection. Resistance Assessment Late blight symptoms were evaluated at 7 and 14 days after inoculation (DAI) by using the modified rating scheme of Saleem et al. (2011). The disease severity index was rated as follows: 0, no visible infection (resistant); 1, a few minute lesions, approximately 10% of the total leaf area is blighted (moderately resistant); 2, approximately 50% of the total leaf area is blighted (susceptible); and 3, most leaves display symptoms, at least 75% of the total leaf area is blighted (severely susceptible). Disease severity was calculated according to the following formula: Disease severity (%) = [number of plants with rating rating score] total number of plants Estimation of the Fruit Weight, Firmness, and Sugar Content of Hybrids Weight of all harvested fruits was determined at 60 days after transplanting 11 NV F 1 hybrids that showed resistance to late blight. The weight of each fruit was estimated by multiplying the average weight by the number of harvested fruits. Fruit firmness and sugar content were measured for 11 NV F 1 hybrids by using an analyzer (EZ test/ce-500n, Shimadzu, Tokyo, Japan) and a pocket refractometer (PAL-1, ATAGO, Tokyo, Japan), respectively. The averages were taken as mean values. Statistical Analysis The data were subjected to analysis of variance (ANOVA) by GLM procedures using SAS software (version 9.1.2, SAS Institute Inc., Cary, NC, USA). Duncan s multiple range tests were used to determine significant differences among the results. The P value of 0.05 was considered to be significant. Results Screening of Hybrids for Resistance to Late Blight The resistance of four parent cultivars against late blight was tested previously (Kim, 2012). Among them, some of the more resistant cultivars in the parent group were Campari, AV104-7, and L3708 with the lowest disease severity ranging from 4.0 to 6.7 at 14 DAI, whereas cultivar 917 showed moderate resistance with a disease severity of 20.0 at 14 DAI. Thus, it was concluded that the four genotypes are useful as parent cultivars for crossing. The resistances of F 2 progenies of cross A (AV107-4 L3708, 204 plants, Table 1) and cross B (Campari 917 L3708, 203 plants, Table 2) were tested. The F 2 progenies varied in their responses to late blight. Fifteen F 2 plants from cross A and thirteen F 2 plants from cross B were resistant. These 28 resistant plants were used to produce F 3 progenies. Five plants derived from each cross were selected for further analysis on the basis of their resistance. Many of the remaining plants 140 (68.6%) of cross A and 166 (81.8%) of cross B, showed moderate resistance similar to that of the parent cultivars in this test. However, 49 progenies from cross A and 24 progenies from cross B were susceptible. Moderately resistant and susceptible plants were not considered for further breeding program. None of the tested F 2 progenies was severely susceptible to late blight. In the screening of F 3 progenies, variable levels of resistance, with disease severity ranging from 3.8 to 14.0 at 14 DAI, were found (Table 3). At 7 DAI, the disease severity in F 3 lines was similar to that in the control line, TGC-88, but significant differences were observed between F 3 and the

3 122 Duck Hwan Park, Yan Zhang, and Byung Sup Kim Table 1. Segregation of resistance to Phytophthora infestans in the F 2 tomato plants from cross A (AV107-4 L3708) in greenhouse Disease rating z No. of F 2 F 2 individual 0 15 y 6, 8, 11, 15, 16, 38, 45, 95, 109, 115, 120, 146, 169, 190, , 2, 3, 4, 5, 7, 9, 10, 12, 13, 14, 17, 18, 19, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 36, 37, 39, 42, 43, 44, 46, 47, 48, 50, 51, 52, 53, 56, 56-1, 57, 58, 60, 60-1, 61, 62, 63, 66, 67, 73, 74, 75, 77, 78, 82, 83, 84, 85, 86, 87, 88, 88-1, 89, 90, 91, 92, 93, 94, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 113, 116, 117, 118, 119, 121, 122, 123, 125, 127, 130, 131, 132, 133, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 147, 148, 149, 150, 154, 155, 158, 160, 161, 164, 167, 168, 170, 171, 175, 176, 177, 178, 179, 180, 181, 183, 185, 186, 187, 188, 189, 193, 195, 195-1, 196, 197, 198, 199, , 22, 23, 33, 34, 35, 40, 41, 49, 54, 55, 59, 64, 65, 68, 69, 70, 71, 72, 76, 79, 81, 111, 112, 114, 124, 126, 128, 129, 134, 139, 146-1, 151, 152, 153, 156, 157, 159, 162, 163, 165, 166, 172, 173, 174, 182, 184, 192, 194 z Disease rating: 0, resistant; 1, moderately resistant; 2, susceptible; and 3, severely susceptible. y Resistant hybrids selected for generation of F 3 lines. Table 2. Segregation of resistance to Phytophthora infestans in the F 2 tomato plants from cross B (Campari 917 L3708) in greenhouse Disease rating z No. of F 2 F 2 individual 0 13 y 1-1, 8, 14-1, 18, 23, 25, 54-1, 57, 73, 75, 76, 80, , 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 19, 19-1, 20, 21, 24, 26, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 70-1, 71, 72, 74, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 93, 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 106, 107, 109, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 143, 143-1, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195 1, 17, 22, 27, 28, 32, 41, 43, 52, 77, 91, 92, 99, 105, 108, 112, 135-1, 142, 145, 146, 161, , 178, 184, z Disease rating: 0, resistant; 1, moderately resistant; 2, susceptible; and 3, severely susceptible. y Resistant hybrids selected for generation of F 3 lines. Table 3. Late blight disease severity in F 3 tomato lines and a standard control line (TGC-88) in greenhouse Hybrid z Disease severity (%) 7 DAI y 14 DAI A6 A16 A38 A120 A146 B8 B23 B54-1 B73 B76 TGC cde x 4.6 bc 2.8 de 3.6 cde 1.4 e 5.8 bc 7.0 b 3.8 cde 4.2 cde 3.8 cde 4.0 cde 4.2 cd 8.0 cd 6.0 cd 3.8 d 14.0 c 8.8 cd 8.0 cd 6.6 cd 9.0 cd 6.0 cd 60.0 a z A, cross A (AV107-4 L3708); and B, cross B (Campari 917 L3708). y DAI, days after inoculation. x Mean separation within columns by Duncan s multiple range test at P control line at 14 DAI. Screening of NV F 1 Hybrids for Disease Resistance and Qualitative Characters To obtain hybrids with stable disease resistance and qualitative characters, such as fruit weight, firmness and sugar content, five F 3 progenies from each cross with three male-sterile cultivars were crossed and the levels of resistance to late blight and the qualitative characters in the progenies were tested. In total, 25 NV F 1 hybrids were developed and evaluated since crosses between F 3 progenies from cross A with Wonung-3 were not successful. Among them, the newly developed hybrids of B23, B54-1, B73, B76, and A38 with Wonung-1 were considered resistant with disease severity ranging from 15 to 33 at 7 DAI (Table 4). The B23, B76, A38, and A120 crosses with Wonung-2, and B23 and B54-1 crosses with Wonung-3 were found to be resistant with disease severity ranging from 13 to 25 and 23 to 24, respectively, and were selected for evaluation of horticultural characteristics (Table 4). Other hybrids were susceptible to late blight and showed over 40% disease severity. In addition, hybrids between each of the three

4 Hort. Environ. Biotechnol. 55(2): Table 4. Late blight disease severity in NV F 1 tomato hybrids and a standard commercial cultivar (Super Doutaelang) in greenhouse Disease severity (%) Hybrid Wonung-1 Wonung-1 Wonung-3 5 DAI z 7 DAI 5 DAI 7 DAI 5 DAI 7 DAI A bc x 77.5 a 42.5 b 61.7 ab ND y ND A b-f 46.0 b-f 25.0 d-g 57.0 bc ND ND A f-j 20.0 i-k 13.6 g-j 24.0 g-k ND ND A bc 54.0 bc 8.0 ij 13.0 k ND ND A b-f 56.0 bc 9.0 h-j 21.3 i-k ND ND B d-h 43.0 b-g 20.0 d-j 45.0 b-f 18.0 e-j 43.0 b-g B f-j 28.8 f-k 19.0 d-j 25.0 g-k 8.0 ij 24.0 g-k B d-h 33.0 d-j 28.0 b-g 48.0 b-e 13.0 g-j 23.0 h-k B d-i 22.5 i-k 6.7 j 30.0 e-k 26.6 c-g 42.0 c-h B j 15.0 jk 6.0 j 15.0 jk 33.0 b-e 50.0 b-d LA b-d 42.0 c-h 13.0 g-j 23.0 h-k 31.0 b-f 39.0 c-i L j 15.0 jk 13.0 g-j 16.0 jk 5.0 j 20.0 i-k Super Doutaelang 66.0 a at 5 DAI 74.2 a at 7 DAI z DAI, days after inoculation. ND, not determined. x Mean separation within DAI by Duncan s multiple range test at P male-sterile cultivars and L3708 as the controls were generated. These hybrids showed the greatest resistance, but produced smaller fruits than developed NV F 1 hybrids. The qualitative characters of these 11 NV F 1 hybrids were also examined. Only two hybrids, cross 1 (Wonung-1 A38) and cross 4 (Wonung-1 B73) had less sugar content than Wonung-2 (Fig. 1). Cross 4 (Wonung-1 B73) and cross 13 (Wonung-2 B23) had lower fruit firmness than Wonung-2. Crosses 3, 8, and 12 (Wonung-1 B76, B54-1, and B23, respectively), 5 and 10 (Wonung-2 A38 and B76, respectively), and 9 (Wonung-3 B54-1) showed low values in fruit firmness or fruit weight. Therefore, these hybrids were not selected for the development of tomato hybrid cultivars. Finally, cross 7 (Wonung-2 A120) and cross 11 (Wonung-3 B23) were chosen for the tomato breeding program in this study (Fig. 1). Discussion The success of a plant breeding program for disease resistance depends on several factors, such as the precision of resistance assessment, identification of resistance markers, and the application of these factors to the development of agronomically superior cultivars (Picó et al., 2000). As the development of resistant cultivars seems to be vital in all crop breeding strategies, field screening methods should prioritize this objective in the case of late blight. Therefore, we believe that our attempts are valuable for developing late blight-resistant and high quality tomato hybrids in Korea by using Campari, AV107-4, L3708, and 917 as parents, which were confirmed to show inheritable resistance in previous work (Kim, 2012). Partial resistance is a desirable trait in plant breeding, since it is often effective across a broad range of pathogen species or strains (Parlevliet, 1979). The use of F 1 plants is one of the means of doing this because F 1 hybrids have several advantages, such as increased yields, increased fruit quality, resistance against biotic and/or abiotic stresses, as well as their ability to provide a quick and convenient way of achieving multiple-desirable characters (Kumer et al., 1998). In our experiments, the F 1 plants were self-pollinated up to the F 3 generation and exhibited increased resistance from F 2 to F 3 progenies, indicating the possibility of resistance alleles accumulating through these generations, although we did not identify the alleles in question in the present study. After the selection of resistant individuals among the F 2 plants, a total of 28 F 3 lines were developed and evaluated for their resistance in the field by natural infection and in the greenhouse by artificial inoculation, but the data are presented for only 5 lines from each cross, because the remaining lines were susceptible and showed mild or moderate disease symptoms on leaves. We focused the trial of NV F 1 hybrids both on improving the high-value horticultural characters and resistance to late blight. This is why we used cherry tomatoes as mother plants. In addition, cherry tomato plants have stronger traits than

5 124 Duck Hwan Park, Yan Zhang, and Byung Sup Kim B23, exhibited the desired positive characteristics for highvalue tomato quality. In conclusion, the NV F 1 hybrids between A120 and Wonung-2, and between B23 and Wonung-3, may serve for the development of a novel commercial tomato cultivar with strong late blight resistance, and could be of high-value for tomato breeding programs. Acknowledgement: This study was supported by a grant (Project No ) from the Screening Center for Disease Resistant Vegetable Crops of TDPAF funded by the MIFAFF of the South Korean government. Literature Cited Fig. 1. Estimated fruit weight (A), firmness (B), and sugar content (C) of 11 NV F1 hybrids with the following crosses: A38 (1), B23 (12), B54-1 (8), B73 (4), and B76 (3) with Wonung-1; A38 (5), A120 (7), B23 (13), and B76 (10) with Wonung-2; B23 (11) and B54-1 (9) with Wonung-3; L3708 (2, 6) with Wonung-1 and 2, respectively, and LA1033 (14) with Wonung-2, as the control test, and C, Wonung-2. Mean separation by Duncan s multiple range test at P other tomatoes (Kim and Mutschler, 2005), which is another reason for us using cherry tomatoes as mother plants. Among the 25 tested NV F 1 hybrids, 11 hybrids manifested significantly positive resistance and 14 hybrids showed significantly negative resistance. This was expected because the mother plants for crossing to make the NV F 1 generation showed a low resistance level to late blight in the preliminary investigation. However, two hybrids, Wonung-2 A120 and Wonung-3 Becktell, M.C., M.L. Daughtre, and W.E. Fry Epidemiology and management of petunia and tomato late blight in the greenhouse. Plant Dis. 89: Fry, W.E. and S.B. Goodwin Resurgence of Irish potato famine fungus. Bioscience 47: Hakiza, J.J The importance of resistance to late blight in potato breeding in Africa, p In: Proceedings of the global initiative on late blight conference, March 16-19, 1999, Quito, Ecuador. (Abstr.) Kim, B.S Evaluation of tomato genetic sources (Solanum spp.) for resistance breeding against late blight. Res. Plant Dis. 18: Kim, M.J. and M.A. Mutschler Transfer to processing tomato and characterization of late blight resistance derived from Solanum pimpinellifolium L. L3708. J. Amer. Soc. Hort. Sci. 130: Ko, W.H An alternative possible origin of the A2 mating type of Phytophthora infestans outside Mexico. Phytopathology 84: Kumer, S., B.S. Vyakaranhal, Y.B. Palled, P.R. Dharmatti, and A.M. Patil Studies on crossing ratio and pollination time in tomato hybrid seed production (Lycopersicon esculentum Mill.). Agric. Sci. 21: Möller, K., M. Dilger, J. Habermeyer, V. Zinkernagel, W.G. Flier, and H. Hausladen Population studies on Phytophthora infestans on potatoes and tomatoes in southern Germany. Eur. J. Plant Pathol. 124: Moreau, P., P. Thoquet, O. Jocelyne, L. Henri, and G. Nigel Genetic mapping of Ph-2, a single locus controlling partial resistance to Phytophthora infestans in tomato. Mol. Plant-Microbe Interact. 4: Mukalazi, J., E. Adipala, T. Sengooba, J.J. Hakiza, M. Olanya, and H.M. Kidanemariam Metalaxyl resistance, mating type and pathogenicity of Phytophthora infestans in Uganda. Crop Prot. 20: Parlevliet, J.E Components of resistance that reduce the rate of epidemic development. Annu. Rev. Phytopathol. 17: Picó, B., A. Sifres, M.J. Diez, and F. Nuez Searching for new resistances to Tomato yellow leaf curl virus within a highly variable wild Lycopersicon genetic pool. Acta Physiol. Planta 22: Saleem, M.Y., K.P. Akhtar, M. Asghar, Q. Iqbal, and A.R. Khan Genetic control of late blight, yield and some yield related traits in tomato (Lycopersicon esculentum Mill.). Pak. J. Bot. 43: Shattock, R Phytophthora infestans: populations, pathogenicity and phenylamides. Pest Manag. Sci. 58: Zhu, G.N., F.X. Huang, L.X. Feng, B.X. Qin, Y.H. Yang, Y.H. Chen, and X.H. Lu Sensitivities of Phytophthora infestans to metalaxyl, cymoxanil, and dimethomorph. Agric. Sci. China. 7: