Differential response of Mi gene-resistant tomato rootstocks to root-knot nematodes (Meloidogyne incognita)

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1 Crop Protection 25 (6) Differential response of Mi gene-resistant tomato rootstocks to root-knot nematodes (Meloidogyne incognita) Jose -Antonio Lo pez-pe rez a,1, Michelle Le Strange b, Isgouhi Kaloshian a, Antoon T. Ploeg a, a Department of Nematology, University of California Riverside, Riverside, CA 92521, USA b UCCE, Tulare County, 4437 S. Laspina Street, Tulare, CA 93274, USA Accepted 15 June 5 Abstract Non-grafted nematode-susceptible tomatoes and tomatoes grafted onto the Mi-gene nematode-resistant rootstock cultivar Beaufort were grown under a range of Meloidogyne incognita population densities in pots in a greenhouse. Under high nematode densities, yields on resistant rootstocks were higher, and galling and root nematode densities at the final harvest were lower. However, galling and nematode levels on resistant rootstocks were still high, and did not correspond to a resistant response. In a second experiment possible reasons for this response, such as virulence of the nematode population used, a low inherent resistance level of the rootstock, or effect of the scion on the rootstock, were evaluated. The results from the second experiment showed that three different M. incognita populations that were used all behaved similar, and therefore the possibility of a virulent population was excluded. There was no influence of the susceptible scion on the resistant rootstock. Two different resistant rootstocks that were used in the second experiment both retained yields under high nematode densities, but exhibited big differences in root galling and final nematode populations. It is concluded that there are big differences between Mi-gene-resistant tomato cultivars as far as nematode host status, and that some should be considered tolerant rather than resistant. The choice of rootstock cultivar can have important consequences for the performance of a subsequent nematode susceptible crop. r 5 Elsevier Ltd. All rights reserved. Keywords: Tomato; Lycopersicon esculentum; Root-knot nematode; Meloidogyne; Rootstock; Grafting 1. Introduction Root-knot nematodes (Meloidogyne spp.) can cause serious yield losses in tomato, Lycopersicon esculentum. Crops grown on sandy soils, the preferred habitat for these nematodes, are most susceptible to nematode damage. In California, fresh market tomatoes are primarily cultivated in the open field with significantly lesser production in greenhouses. However, greenhouse Corresponding author. Tel.: ; fax: address: antoon.ploeg@ucr.edu (A.T. Ploeg). 1 Current address: Centro de Experimentacio n Agraria de Marchamalo, Calle Extramuros, 1918 Marchamalo, Guadalajara, Spain. production is on the rise (Calvin and Cook, 5). Rootknot nematode species that are commonly found in tomato production are M. javanica and M. incognita. Nematode control with methyl bromide soil fumigation was until recently a common practice, but with the withdrawal of methyl bromide, growers are looking for alternative approaches for nematode control. In addition, there is an increasing market for organically grown tomatoes, where the use of chemical pesticides is not an option. The use of root-knot nematode-resistant tomato cultivars is an attractive alternative for nematode management, as their use does not require major adaptations in farming practices. When resistant tomato cultivars with specific required characteristics (e.g. fruit type) are not available, susceptible cultivars can be /$ - see front matter r 5 Elsevier Ltd. All rights reserved. doi:1.116/j.cropro.5.7.1

2 J.-A. López-Pérez et al. / Crop Protection 25 (6) grafted onto nematode-resistant rootstocks. A range of tomato and rootstock cultivars with resistance against M. incognita, M. javanica and M. arenaria are available. Resistance in these tomato cultivars is based on the presence of the single dominant Mi-1 gene that was introgressed into cultivated tomato from a wild relative L. peruvianum (Medina-Filho and Stevens, 198). Mimediated resistance triggers a hypersensitive response leading to cell death soon after nematodes initiate feeding near the vascular bundle (Dropkin, 1969a). Resistant cultivars and rootstocks can be used under a range of environmental conditions and soil types, but the resistance breaks down under high (above 28 1C) soil temperatures (Dropkin,1969b). This study was initiated to test the usefulness of using a nematode-resistant tomato rootstock with a desirable fresh-market tomato scion, grown in a commercial fresh-market tomato greenhouse in southern California on sandy soil with high M. incognita infestation. 2. Material and methods Two consecutive greenhouse experiments were conducted. In the first experiment, tomato cultivar and rootstock cultivar Beaufort (De Ruiter Seeds C/V, Netherlands) were used. is an indeterminate beeftype greenhouse cultivar with no nematode resistance. Beaufort (TmKNVF2Fr) is a rootstock with Mi-gene resistance against the three most common root-knot nematode species. There were three plant treatments: grafted onto Beaufort, grafted onto, and non-grafted. Grafting was done according to seed company instructions (Anonymous, 5). Briefly, an excess number of the rootstock cultivar Beaufort was seeded in a potting mix (Sunshine mix 5, SunGro horticulture Canada Ltd) 1 week prior to seeding, the scion cultivar. Plants were kept in a greenhouse until the 3rd true-leaf stage. Plants of similar size were cleftgrafted and transferred to a clear plastic mist chamber, in a greenhouse delivering 1 s mist/5 min. Over the following week, misting frequency was gradually reduced to zero. Plants were then transferred to a greenhouse bench, maintained at C, and used 1 week later. A race 3 M. incognita population, originally isolated from cotton in the San Joaquin Valley, CA, USA, was maintained and multiplied in a greenhouse on tomato cultivar UC82. Species and race identification were confirmed by iso-zyme electrophoresis and by reproduction on differential hosts (Eisenback and Triantaphyllou, 1991). Nematode inocula consisted of M. incognita eggs that were extracted from tomato roots with a 1% NaOCl solution (Radewald et al., 3). Eggs released from the roots were collected on a 25 mm pore-size sieve and were counted in three.1 ml subsamples. Prior to inoculation, egg concentrations were adjusted to contain 1 2,1 3,1 4,or1 5 eggs/15 ml suspension. No-nematode controls were included. For nematode inoculation, 15 ml of egg suspension was thoroughly mixed with 3.4 kg of a 9:1 mixture of steam-sterilized sand and potting mix, and used to fill 3.5 l pots. Immediately after filling the pots, the non-grafted and grafted seedlings were transplanted. A total of 9 pots were set up: each of the plant treatment (3) and nematode level (5) combinations had 6 replicates. Pots were placed on greenhouse benches according to a randomized block design and watered through an automated drip system. Five days after inoculation, 1 g of a slow release fertilizer (N-P-K: ) was added to each pot. Tomato plants were trained along plastic wires and fruits were harvested at the breaking stage (orange in color) and weighed. Three months after transplanting all remaining fruits were harvested, plants were carefully removed from the pots, and the root systems washed free of soil. Fresh root mass, and degree of root galling (scale 1, ¼ no galls, 1 ¼ 1% galled; Bridge and Page, 198) were determined. Eggs were extracted from the root systems, and counted. In the second experiment, an additional tomato cultivar Hypeel45 (Seminis Inc., CA) was included. Hypeel45 (VF1-2,N,BSK-) is a processing tomato with the Mi-gene resistance. There were five plant treatments: grafted onto Beaufort, grafted onto Hypeel45, non-grafted, non-grafted Beaufort, and non-grafted Hypeel45. Grafting was done as described before. Three different M. incognita populations were used in the second experiment: A population that was extracted from the Mi-gene-resistant Beaufort roots from experiment 1, and was then sub-cultured for 3 months on a fresh Beaufort plant; A population that was extracted from the susceptible roots from experiment 1, and was then sub-cultured for 3 months on a fresh plant; and a race 1 population of M. incognita originally from grape at Coachella, CA, that had been maintained and cultured on tomato cultivar Tropic. Nematodes were extracted from roots and inocula were prepared as described before, but with only one inoculum density of 5, eggs per pot. Each combination of plant treatment (5) and nematode population (3) had five replicates resulting in a total of 75 pots. Experimental design, and data collection (fruits only collected from ) were as described before Data analysis Effects of treatments on root galling, total kg fruit harvested, and nematode populations at harvest were analyzed using ANOVA procedures. The significance of differences within treatments was tested using Duncan s

3 384 ARTICLE IN PRESS J.-A. López-Pérez et al. / Crop Protection 25 (6) test. Analyses were performed using SAS statistical software (SAS Institute, Cary, NC, USA) Detection of Mi gene presence To confirm the genotype of tomato cultivars Beaufort and, four 6 week-old plants of each cultivar were used in PCR analysis. DNA was extracted from each of the 8 root systems by grinding in liquid N 2 and extracting with phenol/chloroform according to established protocols (Sambrook et al., 1989). To determine the presence of Mi gene, a DNA marker REX-1 linked to Mi was amplified using PCR and DNA from both cultivars as described in Williamson et al. (1994). The amplified products were digested with TaqI restriction enzyme and electrophoresed on 1.5% agarose gel. 3. Results 3.1. Experiment 1 Grafting had a significant effect on all measured parameters while the nematode inoculum density had a significant effect on all measured parameters but root mass. Interactive effects of grafting inoculum density were significant for tomato root mass and galling (Table 1). Compared to the non-grafted, grafting onto Beaufort significantly increased the total fruit mass whereas grafting on lowered the fruit mass. grafted onto Beaufort rootstocks also produced more fruits, and reduced the number of nematode eggs produced on the roots (Table 2). The number of fruits produced decreased with each incremental increase of inoculum density, but the average total fruit mass was significantly lower only at the highest inoculum density (data not shown). Where nematodes had been inoculated, tomato plants grafted onto Beaufort roots had significantly less root galling, although levels were still considerable (Table 3). To confirm the root-knot nematode resistance genotypes of Beaufort and tomato cultivars, genotyping was performed using REX-1 marker analysis. As expected, amplification of REX-1 marker using DNA from either cultivar as template resulted in a single DNA fragment of 77 bp (data not shown). Restriction of the amplified PCR products from Beaufort tomato with TaqI enzyme resulted in two fragments of 6 and 17 bp, indicating the presence of the L. peruvianum introgressed DNA, and therefore the Mi gene. No TaqI restriction site was present in the amplified PCR products from tomato samples indicating the absence of L. peruvianum DNA and the Mi gene (data no shown). At the lower ( 1 eggs/pot) inoculum range, fresh root mass of Beaufort rootstocks was higher (Pp:5) than those of roots and rootstocks (data not shown). However, at the higher nematode densities (1, and 1, eggs/pot) the root mass was similar for the three graft treatments (data not shown). Analysis of fruit production over time showed significant differences among the three graft treatments mainly at the higher inoculum densities, and during the end of the harvest period (Figs. 1A C). Generally, at the higher inoculum densities, grafted onto Beaufort continued to produce fruits until the final harvest, whereas non-grafted and grafted onto, ceased to produce fruits towards the end of the experiment (Figs. 1B and C). Table 2 Effect of grafting on average a tomato fruit yield and nematode infestation Graft treatment Number of fruit Total fruit mass (g) 8.3b c 79.6b 1,485,75a onto 7.b 584.9c 1,411,667a onto Beaufort 1.4a 899.3a 84,117b a Yield and nematode infestation averaged over five inoculum densities. Green fruits remaining on plants at final harvest included. b P f : average number of eggs extracted per root system. Nontransformed data shown, statistical analysis was performed on log(p f +1)-transformed data. c Different letters in the same column represent significant differences at the 95% confidence level. P f b Table 1 Significance (P-values) of grafting and nematode inoculum density on tomato plant growth and nematode infestation Factor Number of fruit Total fruit mass Root mass Galling a Log(P f +1) b Grafting o.1 o.1 o.1 o.1 o.1 Inoculum density o.1 o.1.14 o.1 o.1 Grafting Inoculum density o.1 o.1.65 a Root galling index from ¼ no galling to 1 ¼ 1% of root galled. b P f : number of eggs extracted from root systems.

4 J.-A. López-Pérez et al. / Crop Protection 25 (6) Table 3 Effect of grafting and inoculum density on tomato root galling Graft treatment Inoculum density (M. incognita eggs per 1 gallon pot) a a 5.a 5.83a 7.5a 8.67a on a 4.33a 5.5a 6.83a 8.33a on Beaufort a 2.17b 2.17b 3.5b 6.67b 3.2. Experiment 2 The total cumulative fruit yield was not significantly different among the three M. incognita populations. However, the grafting treatments did result in significant differences. Yields from nongrafted were lower (Pp:5) compared to the other two treatments. Yields of tomatoes grafted on the two resistant rootstocks were higher, but not significantly different from each other (/Beaufort /Beaufort / (A) /Beaufort / (B) /Beaufort / (C) Fig. 1. Cumulative fruit yield of non-grafted, grafted onto, and grafted onto Beaufort. (A) without nematodes, (B) with 1 3, and (C) with 1 5 Meloidogyne incognita eggs per 3,5 l pot. Vertical lines represent7standard error. Green fruits remaining at final harvest not included.

5 386 ARTICLE IN PRESS J.-A. López-Pérez et al. / Crop Protection 25 (6) vs. /Hypeel45). The interactive effect of the two treatments (population grafting) was not significant (Table 4). Table 4 Effect of grafting and origin of M. incognita population on tomato fruit yield a (g) Tomato M. incognita population Coachella Beaufort Average non-grafted b b on Beaufort a on Hypeel a Average a Green fruits remaining at final harvest included. b Different letters within the last column represent significant differences at the 95% confidence level. Analyzing the fruit yield over time per population (Figs. 2A C), showed that the M. incognita population from Coachella inhibited fruit production of non-grafted at an early stage. The population originating from resistant Beaufort roots reduced fruit yield of and grafted onto Beaufort significantly more than yield of grafted onto Hypeel45. In pots with the M. incognita population originating from infested susceptible roots, fruit yield of grafted were significantly higher than those of non-grafted from about harvest day 25 onward. The effects of grafting on severity of root galling were similar to effects on fruit yield. Effects of the origin of the M. incognita population or the interactive effect population grafting were not significant (Table 5). The type of rootstock however did cause significant differences in galling. As expected, galling was highest on /Beaufort /Hypeel45 (A) /Beaufort /Hypeel45 (B) /Beaufort /Hypeel45 (C) Fig. 2. Cumulative fruit yield of non-grafted, grafted onto Beaufort, and grafted onto Hypeel45. Inoculated with Meloidogyne incognita eggs per 3,5 l pot. (A) population originating from roots, (B) population originating from Beaufort roots, and (C) population originating from Coachella vineyard. Vertical lines represent7standard error. Green fruits remaining at final harvest not included.

6 J.-A. López-Pérez et al. / Crop Protection 25 (6) Table 5 Effect of grafting and origin of M. incognita population on tomato root galling Grafting Table 6 Effect of grafting and origin of M. incognita population on nematode reproduction a M. incognita population Grafting Coachella Beaufort Average non-grafted 2,11, 3,165, 2,25, 2,58,333a b on Beaufort 2,36, 2,55,7 1,86, 2,256,9a Beaufort non-grafted 3,7, 4,64, 3,45, 3,528,a on Hypeel45 3,13 136,7 111,9 97,361b Hypeel45 non-grafted 24,1 77,8 65,8 53,913b Average roots of non-grafted. Galling on Beaufort roots was significantly lower, but no difference between roots onto which had been grafted and non-grafted Beaufort plants was observed (Table 5). Finally, galling on Hypeel45 roots was much lower, both when used as a rootstock for, and when Hypeel45 was not grafted (Table 5). On average, the Coachella population produced fewer eggs on roots than the populations originating from Beaufort or roots (Table 6). The tomato root type also had a strong effect on nematode reproduction (Table 6). Egg production was high and similar on and on Beaufort roots, but fewer eggs were produced on the Hypeel45 roots (Table 6) There were no interactive effects. 4. Discussion and conclusion M. incognita population Coachella Beaufort Average non-grafted a a on Beaufort b Beaufort non-grafted b on Hypeel c Hypeel45 non-grafted c a Different letters within the last column represent significant differences at the 95% confidence level. 1,518,586b c 1,998,84a 1,547,54a a Number of eggs per root system, statistical analysis on log(x+1) transformed data, untransformed data shown. b Different letters within the last column represent significant differences at the 95% confidence level. c Different letters within the last row represent significant differences at the 95% confidence level. Cultivation of nematode-resistant cultivars has a dual purpose, to avoid crop damage by nematodes, and to reduce nematode population levels. As such, nematoderesistant cultivars can also aid a subsequently grown nematode susceptible crop (Colyer et al., 1998; Hanna, ; Ornat et al., 1997). In our first experiment, grafting-susceptible tomato on the nematode-resistant rootstock Beaufort significantly increased fruit yields compared to non-grafted controls. At harvest, although galling and nematode levels on resistant rootstock Beaufort were lower than on non-grafted susceptible controls, levels were still high. For example, average P f / P i values (final egg density/inoculum density) for the 1 2,1 3,1 4, and 1 5 inoculum densities were 1,291, 812, 67, and 17, respectively, for tomato plants grafted onto resistant Beaufort rootstocks, indicating a strong increase in nematode levels over the duration of the experiment. We speculated that this unexpected result could have been due to a number of factors: (1) The soil temperature during the experiment had been (too) high, resulting in resistance breaking. Nematode resistance mediated by the Mi gene has been reported to break at and above soil temperatures of 28 1C (Dropkin, 1969b). The soil temperature in the pots in experiment 1 ranged between 2 and 24 1C, and therefore the observed high galling and nematode levels on the resistant rootstock can not be attributed to resistance breaking caused by high soil temperatures. (2) The nematode population that was used was virulent on resistant tomato carrying the Mi gene. This was unlikely however because the M. incognita that was used had never before been exposed to resistant tomato. To eliminate this possibility, another M. incognita population (from grape, Coachella) that had never been exposed to tomato carrying the Mi gene was included. Furthermore, to identify the possible selection of virulence in the population used in experiment 1, the population was sub-cultured both on resistant and susceptible tomato and subsequently used in experiment 2. (3) The Beaufort seeds had been mixed up and were in fact of another non-resistant cultivar. This possibility was eliminated, as the presence of the Mi gene was detected by PCR in all of four tested Beaufort plants. (4) The grafting procedure itself, or the presence of a susceptible scion on the resistant rootstock resulted in resistance breaking. Although very unlikely, this hypothesis was tested by including non-grafted Beaufort in the second experiment. Finally, Beaufort was compared to another root-knot nematode resistant tomato cultivar Hypeel45 that also carries the Mi gene. Results with Beaufort in the second experiment confirmed the results from the first experiment. Fruit yields of tomato grafted on this rootstock were higher than those of non-grafted tomato. Galling on Beaufort roots was lower than on, but still high (average gall index 6.1). Irrespective of the nematode source, final nematode populations were also high, both on nongrafted Beaufort and on Beaufort used as a rootstock for, and not different from nematode levels on non-grafted. Thus, the hypothesis that the grafting

7 388 ARTICLE IN PRESS J.-A. López-Pérez et al. / Crop Protection 25 (6) procedure or the susceptible scion had lowered the level of resistance of the Beaufort rootstock was not supported. The three Meloidogyne populations behaved similarly on tomato plants with the different graft combinations. On average, the Meloidogyne population from Coachella resulted in slightly lower nematode levels at harvest, but the fruit yields or galling levels were not affected by the origin of the Meloidogyne population. Thus, there are no indications that the population that was used in experiment 1 was a virulent population with the ability to break resistance mediated by the Mi gene. There were no significant differences in fruit yield between the two nematode-resistant tomato cultivars Hypeel45 and Beaufort when used as rootstocks for. However, nematode data were very different. Galling and final nematode populations were much lower on Hypeel45 than on Beaufort roots. It can therefore be concluded that the Beaufort rootstock rendered susceptible tomatoes to become tolerant to M. incognita infestation, whereas Hypeel45 prevented both nematode damage and a strong nematode build-up. In spite of the presence of the Mi introgressed region in Beaufort, low levels of resistance of Beaufort against M. arenaria have been reported earlier (Graf et al., 1). In those experiments, three different root-knot nematoderesistant rootstocks carrying the Mi gene were tested. The reason for the high level of galling and nematode reproduction on Beaufort, in spite of the presence of the L. peruvianum introgressed region, remains unknown. A possible explanation is mutation(s) in the Mi gene or a gene required in the Mi-mediated resistance pathway. Another reason could be gene inactivation due to DNA methylation as transcriptional regulation due to DNA methylation is well documented. Therefore, it is possible that the Mi gene, or another gene in the Mi-signaling pathway, is not transcribed due to DNA methylation. Indeed a report exists about inactivation of the Mi gene due to DNA methylation in a tomato line identified in a tomato breeding program (Liharska, 1998) Our results have important agronomical implications, as they demonstrate that nematode-resistant tomato rootstocks can be used for grafting desirable tomato scions. Production of the grafted seedlings is simple and success rate is quite high (94% in our experiments) allowing the farmer a wide choice of tomato cultivars for commercial production. The higher cost of purchasing or producing grafted transplants can be more than compensated by increased yields and by savings resulting from a reduction in pesticide use (Besri, 3). However, our results also indicate that the presence of the Mi introgressed region does not necessarily result in root-knot nematode resistance or low post-plant nematode levels. Therefore, tomato cultivars should be carefully selected especially when they will be followed by a nematode susceptible crop. Acknowledgments The authors would like to acknowledge S. Edwards for his expertise and technical assistance, V. Williamson for helpful discussions and Scott Shacklett for allowing us to use his greenhouse for preliminary experiments. We thank De Ruiter Seeds and Seminis Inc. for donating tomato seeds. References Anonymous, 5. Tomato rootstock cultural information. Guidelines for Grafting. De Ruiter Seeds C/V, The Netherlands 4pp. Besri, M., 3. Tomato grafting as an alternative to methyl bromide in Marocco. Proceedings of the 3 Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, CA, USA. Bridge, J., Page, S.L.J., 198. Estimation of root-knot nematode infestation levels on roots using a rating chart. Tropical Pest Manage. 26, Calvin, L., Cook, R., 5. North American greenhouse tomatoes emerge as a major market force. Economic Research Service, USDA. Amber Waves 3, Colyer, P.D., Kirkpatrick, T.L., Vernon, P.R., Barham, J.D., Bateman, R.J., Reducing Meloidogyne incognita injury to cucumber in a tomato-cucumber double-cropping system. J. Nematol. 3, Dropkin, V.H., 1969a. Cellular responses of plants to nematode infections. Ann. Rev. Phytopath. 7, Dropkin, V.H., 1969b. The necrotic reaction of tomatoes and other hosts resistant to Meloidogyne: reversal by temperature. Phytopathology 59, Eisenback, J.D., Triantaphyllou, H.H., Root-knot nematodes: Meloidogyne species and races. In: Nickle, W.R. (Ed.), Manual of Agricultural Nematology. Marcel Decker, New York, NY, USA, pp Graf, V., Augustin, B., Laun, N., 1. Veredelung von Tomatenpflanzen Sicherheit vor Wurzelgallenalchen und Korkwurzelkrankheit [Grafting of tomato plants protection against root gall nematodes and cork root disease]. Gemuse-Munchen 37, Hanna, H.Y.,. Double-cropping muskmelons with nematoderesistant tomatoes increases yield, but mulch color has no effect. Hortscience 35, Liharska, T., Genetic and molecular analysis of the tomato rootknot nematode resistance locus Mi-1. Ph.D. Thesis, Department of Molecular Biology, Wageningen Agricultural University, Wageningen, The Netherlands. Medina-Filho, H.P., Stevens, M.A., 198. Tomato breeding for nematode resistance: survey of resistant varieties for horticulural characterisitcs and genotype of acid phosphates. Acta Horticult. 1, Ornat, C., Verdejo-Lucas, S., Sorribas, F.J., Effect of the previous crop on population densities of Meloidogyne javanica and yield of cucumber. Nematropica 27, Radewald, K.C., Darsow, J., Stangelini, M.E., Becker, J.O., 3. Quantitative comparison of methods for recovery of root-knot nematode eggs from plant roots. Phytopathology 93, S129. Sambrook, J., Fritsch, E.F., Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Country. Williamson, V.M., Ho, J.-Y., Wu, F.F., Miller, N., Kaloshian, I., A PCR-based marker tightly linked to the nematode resistance gene, Mi, in tomato. Theor. Appl. Gen. 87,