Horticultural Science and Technology. August 2019. 509-519
https://doi.org/10.7235/HORT.20190051

MAIN

  • Introduction

  • Materials and Methods

  •   Nematode Isolates

  •   Plant Materials

  •   Detection of Mi Gene

  •   Bioassay

  •   Data Analysis

  • Results

  •   Detection of Mi-1.2 Gene

  •   Bioassay

  • Discussion

Introduction

Tomatoes (Solanum lycopersicum L.), members of the Solanaceae family, are typical components of human nutrition and used extensively in diverse cuisines in the world with annual production of 177 million tons in 2016 (FAO, 2018). Turkey is the fourth largest tomato producer in the world, which accounts for 7.2% (12.7 million tons) of the total annual production in 2016 (FAO, 2018).

Root-knot nematodes (RKN), Meloidogyne spp., are a major threat for tomato production due to their short life cycles and high reproductive rates (Trudgill and Blok, 2001). They have high damage potential and economic impact on tomato production especially in protected cultivation because of intensive cropping with a short fallow period, as well as suitable soil conditions under these production systems that ensure a favorable environment for high nematode population growth on susceptible cultivars (Lopez-Gomez et al., 2015). Therefore, effective nematode control is required to improve crop productivity in these systems. Although nematicides have successfully controlled RKN populations, increasing human and environmental health concerns resulted in the restrictions on broad-spectrum nematicides. These limitations have contributed to an increased interest in the use of resistant tomato cultivars as an alternative control measure (Ornat et al., 2001; Devran et al., 2010). In addition, this increased interest in the use of resistance is because growing resistant plants/cultivars, instead of susceptible ones, does not require any additional farming practices (Lopez-Perez et al., 2006; Cortada et al., 2009; Devran et al., 2010).

RKN resistance in tomato cultivars and rootstocks is mediated by the single dominant Mi-1.2 gene that was introgressed from a wild tomato species, Solanum peruvianum (Smith, 1944). Different molecular markers have been developed to detect the presence of this gene, which is located on the short arm of chromosome 6 (Williamson et al., 1994; El Mehrach et al., 2005; Seah et al., 2007). Furthermore, these markers indicate the Mi allelic conditions (MiMi, Mimi, and mimi) at the gene locus (Cortada et al., 2008; Devran et al., 2013). Plant genetic background has a major effect in the variability of the resistance response to nematodes (Jacquet et al., 2005; Lopez-Perez et al., 2006; Cortada et al., 2009; Verdejo-Lucas et al., 2009). Several authors suggested a possible dosage effect of the Mi-1.2 gene that is more effective against the nematode in homozygous (MiMi) than heterozygous (Mimi) conditions (Tzortzakakis et al., 1998; Jacquet et al., 2005). Additionally, inter- and intra-specific genetic variability of Meloidogyne isolates contributes to the variation of the Mi-1.2 response (Cortada et al., 2009). Therefore, Mi-1.2-mediated resistance is strongly influenced by the interaction between plant genotype and nematode isolate (Jacquet et al., 2005). Another major influence on the performance of the Mi-1.2 gene is temperature. The efficacy of Mi-1.2-mediated resistance is reduced at soil temperatures above 28°C (Dropkin, 1969; Devran et al., 2010). However, daily fluctuations in soil temperatures with intermittent peaks above 28°C did not endanger the resistance (Araujo et al., 1982a; Verdejo-Lucas et al., 2013). The response of Mi-1.2 gene exposed to high temperature was changed according to the length of the high temperature period, heat intensity, or their interaction (Verdejo-Lucas et al., 2013). Furthermore, resistance was affected by the timing of the high temperature period as it was lost when applied at the beginning of the experiment (young plants) shortly before or after nematode inoculation (Dropkin, 1969; Carvalho et al., 2015).

Meloidogyne-resistant tomato cultivars and rootstocks have been widely used in many greenhouses globally. Therefore, it is essential to explore the response of resistant tomato genotypes to RKN species in non-temperature controlled greenhouses for the development of effective control strategies. Although many studies on the response of tomato cultivars and rootstocks carrying the Mi-1.2 gene against different isolates of Meloidogyne species have been reported (Ammati et al., 1986; Tzortzakakis et al., 1998; Ornat et al., 2001; Molinari and Caradonna, 2003; Lopez-Perez et al., 2006; Cortada et al., 2008, Cortada et al., 2009; Devran et al., 2010; Verdejo-Lucas et al., 2013; Carvalho et al., 2015), little is known about the variation in the resistance response of tomato cultivars and rootstocks to Meloidogyne isolates at different growing periods (Cortada et al., 2008, Cortada et al., 2009). The objectives of this study were to identify the Mi-1.2 gene zygosity form in four tomato cultivars and four tomato rootstocks by PCR-based co-dominant SCAR marker Mi23, and to determine the reaction of these tomato genotypes to eight different local isolates of M. arenaria, M. incognita, and M. javanica in pot experiments conducted in a non-temperature controlled greenhouse for short and long growing periods.

Materials and Methods

Nematode Isolates

Eight RKN isolates including four of M. arenaria (Er-4, A-7, Sn-11, and B-17), two of M. incognita (Pr-4 and A-11) and two of M. javanica (Ço-1 and Çr-27) were used in this study. The cultures of these isolates, collected in the commercial greenhouses in the Middle Black Sea Region of Turkey, were initiated from single egg masses and species were identified in a previous study (Aydınlı and Mennan, 2016). These isolates were maintained on nematode-susceptible tomato cv. Falcon in pots. The species identification of these isolates was confirmed using esterase phenotypes (Esbenshade and Triantaphyllou, 1985) before the pot experiments for bioassay were conducted.

Plant Materials

Tomato seedlings were purchased from a commercial supplier of vegetable seedlings (Olympus Fide, Antalya, Turkey). Five tomato cultivars and four rootstocks were used in the experiments (Table 1). Tomato ‘Barbaros’ was used as a nematode-susceptible control and also was grafted onto rootstocks.

Table 1. Origins and resistances of tomato cultivars and rootstocks used in this study

Tomato Seed Company Resistancez
Rootstocks
Arazi Sygenta Seeds HR: ToMV 0-2/Ff A-E/Fol 1-2/For/Va IR: PI/ Ma/Mi/Mj
Beaufort De Ruiter HR: ToMV/Fol:0,1/For/PI/Va/Vd/Ma/Mi/Mj
Comfort Tolya Seeds HR: ToMV/Fol:0,1/Va/Vd/For/Pl/Ma/Mi/Mj
Kingkong Rijk Zwaan HR: ToMV:0-2/Fol:0,1/For/Pl/Va:0/Vd:0 IR: Ma/Mi/Mj
Cultivars
Adamset Sygenta Seeds HR: TSWV/For/Va IR: Ma/Mi/Mj
Alsancak Yüksel Seeds HR: ToMV/Va/Vd/Fol: 0-1/Cf-5 IR: Ma/Mi/Mj
Barbaros Seven Brothers Seeds ToMV, Va, Fol: 0,1, TSWV
Crisol Semillas Fitó HR: ToMV/TSWV/Fol:0,1/Vd/Mi
Esin Zeraim Gedera HR: Vd/Fol/ToMV/TSWV IR: Ma/Mi/Mj
zInformation was obtained from product catalogues or the website of the seed companies. HR: high resistance; IR: intermediate resistance; ToMV: tomato mosaic virus; TSWV: tomato spotted wilt virus; Va: Verticillium albo-altum; Vd: V. dahliae; Fol:0,1: Fusarium oxysporum race 0 and 1; For: Fusarium oxysporum f. sp. radicis-lycopersici; Cf: Cladosporium fulvum; Ff: Fulvia fulva; PI: Pyrenochaeta lycopersici; Ma: Meloidogyne arenaria; Mi: M. incognita; Mj: M. javanica.

Detection of Mi Gene

To assess whether test plants carried the Mi-1.2 resistance gene and the homozygous or heterozygous condition for the Mi locus, a PCR study was performed using SCAR primers Mi23F (5’-TGG AAA AAT GTT GAA TTT CTT TTG-3’) and Mi23R (5’- GCA TAC TAT ATG GCT TGT TTA CCC-3’) specifically designed for this gene in tomato (Seah et al., 2007). DNA was extracted from young leaf tissue and root tissue of non-grafted and grafted seedlings, respectively, using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions.

PCR reactions were performed in a final volume of 25 µL containing 20 ng template DNA, 1X Taq buffer with KCl, 2 mM MgCl2, 0.2 mM dNTPs, 0.4 µM of each primer, and 1.25 U of Taq DNA Polymerase (Thermo Scientific, Waltham, MA, USA). PCR was conducted with a T-100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) and amplification conditions comprised an initial denaturation step at 94°C for 3 min, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C, with a final extension for 8 min at 7°C. PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide and photographed by the gel documentation system, G:BOX F3 (Sygene, Cambridge, UK).

Bioassay

Experiments were conducted to evaluate the resistance response of tomato cultivars and rootstocks to different Meloidogyne species and isolates in short (8 weeks) and long (16 weeks) growing periods in a non-temperature controlled greenhouse (17-44°C). Seedlings were transplanted singly into plastic pots containing approximately 500 cm3 and 1,500 cm3 of sterilized sandy soil for short period and long period experiments, respectively, and allowed to grow for 1 week before nematode inoculation. Eggs of Meloidogyne isolates were obtained by shaking the infected tomato roots in a 0.5% NaOCl solution for 3 min (Hussey and Barker, 1973). The suspension containing the eggs and roots was poured onto 200 and 500 mesh sieves, and eggs were collected from the 500 mesh sieve. Egg suspensions were quantified and used as inocula. Plants were inoculated with 3,000 eggs (Pi) per plant by pipetting the egg suspension into three holes made in the soil around the plant (28 April). Each nematode isolate-plant genotype combination was replicated five times for both experiments. Pots were placed on greenhouse benches according to a randomized block design. The experiments were terminated 8 weeks (22 June) and 16 weeks (17 August) after nematode inoculation for the short and long growing periods, respectively. The root systems were removed from the pots, washed with tap water, and rated for gall index (GI) using a 0-10 scale (Bridge and Page, 1980). Then, the roots were macerated in a blender with a 1% NaOCl solution three times for 15 seconds with 30 seconds intervals, the suspension poured onto 200 and 500 mesh sieves, and eggs were collected from the 500 mesh sieve for determining the final population density (Pf) of each combination. The reproduction factor (Rf = Pf/Pi) and reproduction index (RI = Pf on the resistant cultivar or rootstocks/Pf on susceptible cultivar x 100) were calculated (Ornat et al., 2001; Cortada et al., 2008). The resistance level of tomato cultivars and rootstocks carrying the Mi-1.2 gene to each RKN isolate was categorized according to the RI as highly resistant (RI < 10%), moderately resistant (10 ≤ RI < 50%), or susceptible (RI ≥ 50%) (Cortada et al., 2009).

Data Analysis

Host reaction of tomato genotypes to each RKN isolate in the short and long growing periods was analyzed separately. Data on GI and Rf were log-transformed [log10(x+1)] prior to analysis, and then subjected to analysis of variance (ANOVA). The Tukey HSD was used to compare means when the ANOVA analysis was significant (p < 0.05). Analyses were performed using SAS statistical software (SAS Institute, Cary, NC, USA).

Results

Detection of Mi-1.2 Gene

In the amplified PCR products from DNA of all rootstocks except ‘Comfort’, a single band of 380 bp was obtained, indicating the homozygous resistant form (MiMi) at the Mi locus (Fig. 1). The cultivars ‘Alsancak’, ‘Crisol’, ‘Esin’, ‘Adamset’, and rootstock ‘Comfort’ gave two bands of 380 and 430 bp that were classified as heterozygous resistant (Mi/mi). ‘Barbaros’ used as a susceptible control displayed a single DNA fragment with 430 bp, confirming the absence of the Mi-1.2 gene (mi/mi).

http://static.apub.kr/journalsite/sites/kshs/2019-037-04/N0130370409/images/HST_37_04_09_F1.jpg
Fig. 1.

Detection of the Mi-1.2 gene in tomato cultivars (lanes 1-5, 9) and rootstocks (lanes 6-8) using primers Mi23F and Mi23R. M: Molecular marker with 100 bp, 1: ‘Adamset’, 2: ‘Alsancak’, 3: ‘Crisol’, 4: ‘Esin’, 5: ‘Comfort’, 6: ‘Arazi’, 7: ‘Beaufort’, 8: ‘Kingkong’, 9: ‘Barbaros’, W: water as a negative control.

Bioassay

As expected, the highest GI values in both growing periods were recorded on the susceptible control ‘Barbaros’, ranging from 6.0 (M. javanica Çr-27) to 7.0 (M. javanica Ço-1) in the short period experiment and from 7.4 (M. arenaria Sn-11 and B-17) to 8.2 (M. incognita Pr-4) in the long period experiment (Table 2). In the short period experiment, GI values were lower on tomato genotypes with Mi-1.2 gene than on the susceptible control ‘Barbaros’ (p ˂ 0.05), with the exception of the resistant ‘Crisol’ inoculated with M. incognita A-11 that had a GI of 5.4 and did not differ from the susceptible control (p > 0.05). Moreover, resistant tomato cultivars had higher galling rates than resistant rootstocks in the short period experiment when inoculated with M. incognita A-11 (p < 0.05). Similarly, GI values on all resistant rootstocks except ‘Kingkong’ were lower than on the resistant cultivars inoculated with M. arenaria Er-4 in this experiment (p < 0.05), but not in the long period experiment. There were no differences (p > 0.05) in galling rate among resistant cultivars for each nematode isolate when resistant genotypes were inoculated with M. arenaria Sn-11 or M. incognita Pr-4 in the short growing period and M. arenaria A-7, B-17 or M. javanica Ço-1 in the long growing period.

Table 2. Gall index (GI)z of Meloidogyne arenaria (Er-4, A-7, Sn-11, B-17), M. javanica (ço-1, çr-27), and M. incognita (Pr-4, A-11) on tomato cultivars and rootstocks in pot experiments conducted in a non-temperature controlled greenhouse for short (8 weeks) and long (16 weeks) growing periods after inoculation with 3,000 eggs per plant

Tomato M. arenariaM. javanicaM. incognita
Er-4 A-7 Sn-11 B-17 ço-1 çr-27 Pr-4 A-11
Short period
Rootstock
Arazi 0.20 ± 0.20ycx 0.20 ± 0.20 c 0.40 ± 0.24 b 0.20 ± 0.20 c 1.20 ± 0.20 bc 0.60 ± 0.24 c 0.40 ± 0.24 b 1.40 ± 0.24 d
Beaufort 0.20 ± 0.20 c 0.60 ± 0.24 bc 0.40 ± 0.24 b 0.60 ± 0.24 bc 0.40 ± 0.24 c 1.40 ± 0.24 bc 0.80 ± 0.37 b 0.60 ± 0.24 e
Comfort 0.20 ± 0.20 c 0.20 ± 0.20 c 0.40 ± 0.24 b 0.20 ± 0.20 c 0.60 ± 0.24 bc 1.20 ± 0.20 bc 1.00 ± 0.00 b 1.00 ± 0.00 de
Kingkong 0.60 ± 0.24 bc 0.60 ± 0.24 bc 0.40 ± 0.24 b 0.40 ± 0.24 bc 0.40 ± 0.24 c 1.40 ± 0.24 bc 0.80 ± 0.37 b 1.40 ± 0.24 d
Cultivar
Adamset 1.00 ± 0.00 b 1.00 ± 0.31 bc 1.40 ± 0.24 b 0.80 ± 0.20 bc 1.40 ± 0.24 bc 2.00 ± 0.31 b 0.60 ± 0.24 b 4.20 ± 0.20 bc
Alsancak 1.40 ± 0.24 b 1.20 ± 0.20 b 1.20 ± 0.20 b 0.20 ± 0.20 c 0.40 ± 0.24 c 0.40 ± 0.24 c 0.40 ± 0.24 b 2.80 ± 0.20 c
Crisol 1.40 ± 0.24 b 1.60 ± 0.24 b 1.40 ± 0.24 b 1.60 ± 0.40 b 1.60 ± 0.24 b 2.40 ± 0.24 b 1.00 ± 0.00 b 5.40 ± 0.24 ab
Esin 1.00 ± 0.00 b 0.80 ± 0.20 bc 0.80 ± 0.37 b 0.20 ± 0.20 c 0.40 ± 0.24 c 1.40 ± 0.24 bc 1.40 ± 0.24 b 3.60 ± 0.24 bc
Barbaros 6.60 ± 0.24 a 6.40 ± 0.40 a 6.40 ± 0.24 a 6.40 ± 0.40 a 7.00 ± 0.31 a 6.00 ± 0.31 a 6.60 ± 0.24 a 6.40 ± 0.24 a
Long period
Rootstock
Arazi 2.40 ± 0.24 bc 1.40 ± 0.24 b 1.40 ± 0.24 c 1.60 ± 0.24 b 2.40 ± 0.24 b 1.80 ± 0.20 e 2.40 ± 0.24 d 5.00 ± 0.00 c
Beaufort 2.20 ± 0.37 bc 2.40 ± 0.50 b 1.80 ± 0.20 bc 2.00 ± 0.31 b 2.00 ± 0.00 b 3.20 ± 0.37 cd 3.00 ± 0.31 cd 1.60 ± 0.24 d
Comfort 1.60 ± 0.24 c 1.60 ± 0.24 b 2.00 ± 0.31 bc 1.80 ± 0.20 b 2.00 ± 0.31 b 2.40 ± 0.24 cd 3.80 ± 0.37 bc 4.40 ± 0.24 c
Kingkong 3.00 ± 0.31 b 2.20 ± 0.48 b 2.00 ± 0.00 bc 1.80 ± 0.20 b 1.40 ± 0.24 b 3.40 ± 0.40 cd 3.40 ± 0.24 bcd 4.80 ± 0.20 c
Cultivar
Adamset 1.80 ± 0.20 bc 2.20 ± 0.37 b 2.40 ± 0.40 bc 1.40 ± 0.24 b 2.40 ± 0.24 b 4.20 ± 0.37 bc 2.60 ± 0.24 cd 5.20 ± 0.20 c
Alsancak 2.20 ± 0.20 bc 2.20 ± 0.20 b 2.40 ± 0.24 bc 1.20 ± 0.20 b 1.40 ± 0.24 b 1.40 ± 0.24 e 2.60 ± 0.24 cd 5.40 ± 0.24 c
Crisol 2.40 ± 0.24 bc 2.60 ± 0.24 b 2.80 ± 0.37 b 2.40 ± 0.50 b 2.20 ± 0.20 b 4.60 ± 0.24 b 3.00 ± 0.31 cd 6.80 ± 0.37 b
Esin 2.20 ± 0.20 bc 1.80 ± 0.20 b 1.80 ± 0.20 bc 1.20 ± 0.20 b 1.40 ± 0.24 b 2.20 ± 0.20 de 4.40 ± 0.24 b 5.40 ± 0.24 c
Barbaros 7.80 ± 0.37 a 7.80 ± 0.37 a 7.40 ± 0.24 a 7.40 ± 0.24 a 7.60 ± 0.24 a 8.00 ± 0.31 a 8.20 ± 0.37 a 8.00 ± 0.31 a
zGI was based on a scale from 0 (none) to 10 (dead plants).
yValues were transformed [log10(x + 1)] before analysis. Data represent mean of five replicates ± standard errors. Each growing period was subjected separately to analysis for each nematode isolate.
xValues in the same column sharing the same letter are not significantly different according to Tukey's HSD test at p < 0.05.

Similar to galling rates, the highest reproduction rates of nematode isolates were detected on the susceptible ‘Barbaros’ (p < 0.05) (Table 3). In the short period experiment, only M. incognita A-11 had higher Pf values than Pi on the resistant cultivars with Rf ranging from 1.59 to 2.46 and these values on the resistant cultivars were significantly higher than on the resistant rootstocks (p < 0.05). Similarly, irrespective of the statistical significance, egg productions of this nematode isolate on the resistant cultivars were higher than on the rootstocks in the long period experiment. For all nematode isolate-plant genotype combinations, Rf values on resistant cultivars and rootstocks were similar when inoculated with M. arenaria Er-4 or M. incognita Pr-4 for short period and M. arenaria Sn-11 for the long period (p > 0.05). Although Pf value of M. javanica Çr-27 on resistant cultivar Crisol was close to Pi value in the short period experiment, it was 5.8-fold higher than Pi in the long period experiment. In the long period experiment, of the resistant cultivars and rootstocks inoculated with M. javanica Çr-27 and M. incognita A-11, ‘Crisol’ supported more nematode reproduction than the others, whereas ‘Esin’ resulted in the highest Rf of M. incognita Pr-4 (p < 0.05). The lowest Rf value of M. incognita A-11 in the long period experiment was detected on the rootstock Beaufort, with a Rf of 0.5 (p < 0.05), whereas Rf values of the isolate on other resistant genotypes ranged from 2.82 to 11.09.

Table 3. Reproduction rate (Rf)z of Meloidogyne arenaria (Er-4, A-7, Sn-11, B-17), M. javanica (ço-1, çr-27), and M. incognita (Pr-4, A-11) on tomato cultivars and rootstocks in pot experiments conducted in a non-temperature controlled greenhouse for short (8 weeks) and long (16 weeks) growing periods after inoculation with 3,000 eggs per plant

Tomato M. arenariaM. javanicaM. incognita
Er-4 A-7 Sn-11 B-17 ço-1 çr-27 Pr-4 A-11
Short period
Rootstock
Arazi 0.04 ± 0.04ybx 0.04 ± 0.04 bc 0.03 ± 0.02 c 0.01 ± 0.01 c 0.22 ± 0.06 bc 0.04 ± 0.01 d 0.09 ± 0.06 b 0.18 ± 0.04 d
Beaufort 0.04 ± 0.04 b 0.11 ± 0.04 bc 0.05 ± 0.04 c 0.07 ± 0.03 c 0.06 ± 0.04 c 0.34 ± 0.08 cd 0.14 ± 0.06 b 0.07 ± 0.03 d
Comfort 0.03 ± 0.03 b 0.02 ± 0.02 c 0.07 ± 0.04 c 0.02 ± 0.02 c 0.12 ± 0.05 bc 0.28 ± 0.02 cd 0.21 ± 0.04 b 0.09 ± 0.00 d
Kingkong 0.17 ± 0.08 b 0.10 ± 0.04 bc 0.02 ± 0.01 c 0.06 ± 0.03 c 0.04 ± 0.03 c 0.37 ± 0.11 cd 0.17 ± 0.09 b 0.16 ± 0.03 d
Cultivar
Adamset 0.13 ± 0.03 b 0.27 ± 0.07 bc 0.33 ± 0.13 bc 0.12 ± 0.03 c 0.33 ± 0.07 bc 0.83 ± 0.17 bc 0.08 ± 0.02 b 2.22 ± 0.34 bc
Alsancak 0.15 ± 0.02 b 0.37 ± 0.06 bc 0.28 ± 0.06 bc 0.02 ± 0.02 c 0.03 ± 0.02 c 0.03 ± 0.02 d 0.04 ± 0.02 b 1.59 ± 0.13 c
Crisol 0.41 ± 0.11 b 0.55 ± 0.15 b 0.49 ± 0.15 b 0.68 ± 0.16 b 0.44 ± 0.11 b 1.03 ± 0.10 b 0.18 ± 0.04 b 2.46 ± 0.13 b
Esin 0.10 ± 0.00 b 0.09 ± 0.06 bc 0.18 ± 0.07 bc 0.01 ± 0.01 c 0.08 ± 0.05 c 0.27 ± 0.07 cd 0.44 ± 0.10 b 1.61 ± 0.14 c
Barbaros 39.20 ± 5.96 a 31.60 ± 5.27 a 43.60 ± 4.11 a 26.20 ± 4.61 a 40.40 ± 4.36 a 29.20 ± 4.38 a 33.60 ± 5.17 a 20.50 ± 1.77 a
Long period
Rootstock
Arazi 1.05 ± 0.09 b 0.26 ± 0.06 d 0.40 ± 0.06 b 0.42 ± 0.10 bcd 1.19 ± 0.21 b 0.62 ± 0.09 ef 0.92 ± 0.10 d 5.13 ± 0.40 cd
Beaufort 0.92 ± 0.09 b 0.89 ± 0.28 bcd 0.51 ± 0.10 b 0.66 ± 0.13 bc 0.52 ± 0.06 bc 1.24 ± 0.21 de 1.10 ± 0.23 cd 0.50 ± 0.11 f
Comfort 0.31 ± 0.08 d 0.29 ± 0.07cd 0.61 ± 0.08 b 0.37 ± 0.07 bcd 0.59 ± 0.12 bc 1.02 ± 0.07 de 1.82 ± 0.22 c 2.82 ± 0.28 e
Kingkong 1.28 ± 0.21 b 0.63 ± 0.18 bcd 0.78 ± 0.06 b 0.70 ± 0.11 b 0.44 ± 0.10 c 1.75 ± 0.30 d 1.15 ± 0.05 cd 4.51 ± 0.54 d
Cultivar
Adamset 0.48 ± 0.07 cd 0.72 ± 0.16 bcd 1.03 ± 0.38 b 0.35 ± 0.06 bcd 1.20 ± 0.22 b 3.28 ± 0.34 c 0.97 ± 0.13 d 6.93 ± 0.18 c
Alsancak 0.38 ± 0.05 cd 0.92 ± 0.13 bc 1.11 ± 0.19 b 0.18 ± 0.01 d 0.42 ± 0.13 c 0.38 ± 0.10 f 0.89 ± 0.09 d 6.56 ± 0.26 cd
Crisol 0.76 ± 0.05 bc 1.24 ± 0.12 b 1.06 ± 0.13 b 0.81 ± 0.20 b 0.88 ± 0.13 bc 5.83 ± 0.58 b 1.26 ± 0.14 cd 11.09 ± 0.88 b
Esin 0.38 ± 0.07 cd 0.42 ± 0.09 cd 0.53 ± 0.12 b 0.23 ± 0.03 cd 0.44 ± 0.16 c 0.95 ± 0.15 de 3.24 ± 0.30 b 7.21 ± 0.77 c
Barbaros 54.74 ± 2.18 a 57.26 ± 2.97 a 67.61 ± 4.37 a 49.98 ± 2.90 a 62.17 ± 3.76 a 61.11 ± 5.42 a 61.58 ± 3.93 a 43.04 ± 3.55 a
zRf = Final population density / Initial population density.
yValues were transformed [log10(x + 1)] before analysis. Data represent mean of five replicates ± standard errors. Each growing period was subjected separately to analysis for each nematode isolate.
xValues in the same column sharing the same letter are not significantly different according to Tukey's HSD test at p < 0.05.

All cultivars and rootstocks carrying the Mi-1.2 gene in both growing periods responded highly resistant (RI ˂ 10%) to RKN isolates tested except for M. incognita A-11 (Fig. 2). Of the all resistant genotypes inoculated with M. incognita A-11, in both growing periods, ‘Crisol’ (RI = 12.01% and 26.11%) and ‘Adamset’ (RI = 10.86% and 16.11%) were moderately resistant, whereas ‘Beaufort’ (RI = 0.38% and 1.17%) and ‘Comfort’ (RI = 0.44% and 6.57%) were highly resistant. Although ‘Alsancak’ (RI = 7.77%), ‘Esin’ (RI = 7.89%), ‘Arazi’ (RI = 0.91%), and ‘Kingkong’ (RI = 0.82%) were highly resistant to M. incognita A-11 in the short growing period, these genotypes showed reduced resistance and responded as moderately resistant to the nematode isolate in the long growing period.

http://static.apub.kr/journalsite/sites/kshs/2019-037-04/N0130370409/images/HST_37_04_09_F2.jpg
Fig. 2.

Reproduction index (RI) of Meloidogyne arenaria (Er-4, A-7, Sn-11, B-17), M. javanica (Ço-1, Çr-27), and M. incognita (Pr-4, A-11) on resistant tomato cultivars (‘Alsancak’, ‘Crisol’, ‘Esin’, and ‘Adamset’) and rootstocks (‘Comfort’, ‘Arazi’, ‘Beaufort’, and ‘Kingkong’) in pot experiments conducted in a non-temperature controlled greenhouse for (A) short (8 weeks) and (B) long (16 weeks) growing periods after inoculation with 3,000 eggs per plant. RI: eggs per plant on a resistant tomato divided by eggs per plant on susceptible control×100.

Discussion

Several molecular markers were developed to detect the presence of the Mi-1.2 gene in tomato, which confers resistance to M. arenaria, M. javanica, and M. incognita (Williamson et al., 1994; El Mehrach et al., 2005; Seah et al., 2007). Molecular markers successfully used in tomato breeding also enable determination of whether the Mi-1.2 gene is homozygous or heterozygous (Cortada et al., 2008; Devran et al., 2013). Of the molecular markers related to the Mi-1.2 gene, REX-1 has been widely used but some studies revealed that this marker gave false positive results in plants carrying the Ty-1 gene, which provides resistance to tomato yellow leaf curl virus (TYLCV) because both the Mi-1.2 gene and the Ty-1 gene are located on chromosome 6 and are very close to each other (El Mehrach et al., 2005; Seah et al., 2007; Devran et al., 2013). Therefore, the marker REX-1 could not be used for screening of the Mi-1.2 gene in tomato plants carrying the Ty-1 gene (El Mehrach et al., 2005; Devran et al., 2013). Moreover, the amplified products of the REX-1 marker have to be digested with a restriction enzyme (TaqI) (Williamson et al., 1994). Alternatively, PMi12 and Mi23 markers can be used, which are reliable markers, require no restriction digestion, and allow differentiation between homozygous and heterozygous resistant genotypes (El Mehrach et al., 2005; Seah et al., 2007; Devran et al., 2013). In this study, we preferred to use the Mi23 marker because the PMi12 marker produced additional bands in some of the tomato lines tested by Devran et al. (2013). Our Mi23 marker analysis was in accordance with previous reports regarding the resistant genotypes ‘Beaufort’ (Cortada et al., 2008), ‘Alsancak’, and ‘Esin’ (Devran et al., 2013).

Seventy-two nematode isolate-plant genotype combinations were tested in both short and long period experiments to evaluate the resistance level of tomato cultivars and rootstocks against different RKN isolates. RI values on resistant genotypes were higher in the long growing period than the short growing period, irrespective of the statistical significance. A likely reason for the differential RI values on the resistant genotype between the two experimental periods is the temperature since the experiments were performed in a greenhouse with a non-controlled temperature environment. During the long period experiment, elevated air temperatures above 28°C were registered, providing conditions that could break the resistance mediated by the Mi-1.2 gene (Dropkin, 1969; Ammati et al., 1986; Devran et al., 2010). Verdejo-Lucas et al. (2013) stated that phenotypic expression of the Mi-1.2 gene was not compromised under daily temperature fluctuations and intermittent peaks above 28°C, and the stability of the Mi-1.2 gene was affected by heat intensity, the length of the heated period, or their interaction. A constant temperature of 32°C for 2-3 days was at least required to overcome the resistance (Dropkin, 1969; Araujo et al., 1982b). In this study, none of the resistant genotypes displayed susceptible response when inoculated with any nematode isolates. Moreover, the highly resistant response of the plant genotypes carrying the Mi-1.2 gene did not change, with the exception of those inoculated with M. incognita A-11, despite the observation that the RI values of these genotypes were higher depending on the experimental periods. Thus, in this study, the nematode population is more effective than the temperature in terms of influencing the category of nematode resistance in the tested cultivars and rootstocks. Of the tested nematode isolates, only M. incognita A-11 caused changes in the relative levels of resistance in all plant genotypes carrying the Mi-1.2 gene, except rootstocks ‘Beaufort’ and ‘Comfort’, in the long growing period and ‘Alsancak’, ‘Esin’, ‘Arazi’, and ‘Kingkong’ exhibited reduced resistance, responding as moderately resistant. The reduced resistance response of these genotypes to this nematode isolate may be based on the existence of nematodes with different degrees of virulence within the population used for inoculation (Molinari and Caradonna, 2003; Castagnone-Sereno et al., 2007). Previously, M. incognita A-11, unlike the other nematode isolates tested in the present study, showed RI higher than 10% (RI=15.6), indicating partial virulence of the isolate (Aydınlı, 2014). Therefore, the differential resistance levels of some genotypes against nematode isolates are likely related to nematode virulence. Even if virulent individuals in Pi were present at a low frequency, the rates of virulence will gradually increase during a prolonged growing period of plants. In contrast, the rootstocks ‘Beaufort’ and ‘Comfort’ were classified as highly resistant against M. incognita A-11 in the long growing period. This could be due to the plant genetic background, which might contribute to the stability of Mi-1.2 resistance in the presence of a partially virulent population. On the other hand, the rootstock ‘Beaufort’ was classified as susceptible to M. incognita (Lopez-Perez et al., 2006) and M. javanica (Cortada et al., 2008, Cortada et al., 2009) in previous studies. The response of ‘Beaufort’ to a Mi-avirulent population of M. javanica was susceptible after both one (63 days post-inoculation) and two nematode generations (132 days post-inoculation) (Cortada et al., 2008). However, Cortada et al. (2009) reported that the resistance level of ‘Beaufort’ varied depending on the Meloidogyne isolates, and the response of the rootstock resulted in classification as highly resistant to M. arenaria (MA-68) and M. incognita (MI-CROS), moderately resistant to M. incognita (MI-ALM), and susceptible to two M. javanica (MJ-IBIZA and MJ-05). Additionally, the rootstock ‘Beaufort’ displayed high resistance to M. incognita race 2 at constant 24°C, but moderate resistance at constant 32°C (Devran et al., 2010). Our study indicated that rootstocks ‘Beaufort’ and ‘Comfort’ could be used as an alternative to other resistant genotypes tested in this study if the emergence of virulent nematode populations in infested fields occurs.

It is well documented that the resistance provided by the Mi-1.2 gene could be affected by nematode population (Ornat et al., 2001; Cortada et al., 2008, Cortada et al., 2009), plant genetic background (Tzortzakakis et al., 1998; Jacquet et al., 2005; Lopez-Perez et al., 2006; Verdejo-Lucas et al., 2009), and temperature (Dropkin, 1969; Araujo et al., 1982a; Araujo et al., 1982b; Ammati et al., 1986; Devran et al., 2010; Verdejo-Lucas et al., 2013; Carvalho et al., 2015). However, there were only limited studies on the resistance response of tomato cultivars and rootstocks with the Mi-1.2 gene to different isolates of RKN depending on the duration of the experiment (Cortada et al., 2008, Cortada et al., 2009). The present study confirms the previous studies and reveals that the relative resistance levels of cultivars and rootstocks could vary according to RKN isolates or the length of the growing period. The differential response of the resistant genotype to Meloidogyne isolates may arise from the proportion of virulent individual nematodes within a population (Molinari and Caradonna, 2003; Castagnone-Serona et al., 2007). Therefore, the characteristics of the nematode population should be taken into account in the choice of tomato cultivars and rootstocks for site-specific management of RKN. Moreover, in order to determine the suitable resistant genotype for the growing area where there may be a virulent population, it may be necessary to assess a long growing period, which allows the detection of population increases and changes in the relative levels of resistance in plant genotypes.

Acknowledgements

This study was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project No. 111O793). The authors wish to thank Mr. Oğuzcan Ocaklı and Ms. Asuman Mutlu (Ondokuz Mayıs University, Samsun, Turkey) for the technical assistance, Prof. Dr. Zübeyir Devran (Akdeniz University, Antalya, Turkey) for reading of the manuscript, and Prof. Dr. Isgouhi Kaloshian (University of California, Riverside, USA) for editing the English.

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