Degree of resistance of Solanum torvum cultivars to Mi-1.2-virulent and avirulent isolates of Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne luci

Abstract Root-knot nematodes (RKN) cause yield losses in eggplant-growing areas. There are no known varieties of eggplant (Solanum melongena L.) that are resistant to RKNs. However, the wild relative of eggplant, S. torvum (Sw.), provides resistance to some RKN species and is used as a rootstock for cultivated eggplants. Therefore, determination of the reproductive capacity of nematodes on eggplant rootstocks developed from S. torvum is required for effective management of RKNs that are widely present in vegetable growing areas. In the present study, the degree of reproduction of Mi-1.2-virulent and avirulent isolates of M. incognita, M. javanica, and M. luci on eggplant rootstocks, Hawk and Boğaç, was evaluated in a plant growth chamber. Hawk and Boğaç were resistant (< 10 egg masses per whole root system) to all avirulent and virulent isolates of M. incognita, M. javanica, and M. luci. This study is the first report on the resistance of S. torvum to virulent isolates of M. luci. Results indicate that S. torvum offers broad-spectrum resistance against RKNs.

Eggplant (Solanum melongena L.) is a member of the Solanaceae family and has wide genetic variation in shape, colour and size (Daunay et al., 2001). In 2019, eggplant was grown in 1.8 M hectares, with a total production of 55 M tons worldwide. Turkey is the world's fourth largest eggplant producer after China, India, and Egypt, with an annual production of 8.2 M tons (FAO, 2021). Root-knot nematodes (RKNs) are one of the most important pathogens affecting eggplant. The use of plants resistant to RKNs is the most effective and environment-friendly tactic for management of this pathogen (Seid et al., 2015). Although there are no resistant cultivars of S. melongena, some wild eggplant species (S. torvum) have been found to be resistant to some RKNs (Ali et al., 1992;Mattos et al., 2011;Rahman et al., 2002). However, resistant hybrids of these wild species with cultivars have not been developed because of crossincompatibility between the phylogenetically distant S. torvum and cultivated S. melongena (Daunay et al., 2001;McCammon and Honma, 1983). Grafting with resistant rootstocks is an additional technique that can alleviate the problem of soil borne disease. Solanum torvum has been employed as a rootstock to control RKNs in eggplant cultivation (Uehara et al., 2016(Uehara et al., , 2017, but it has variable responses to the major RKNs. Meloidogyne arenaria (A2-J) (Chitwood, 1949;Neal, 1889) produced large numbers of egg masses on S. torvum root stock cv. Tonashimu, but M. arenaria (A2-O) and M. incognita produced only a few egg masses (Uehara et al., 2017). A study conducted by Garcia-Mendivil et al. (2019) showed that S. torvum rootstock cv. Brutus, Espina, Salutamu and Torpedo were resistant to M. incognita isolates and varied from highly resistant to susceptible to Mi-1.2 (a)virulent isolates of M. javanica (Chitwood, 1949;Treub, 1885) from Spain. In other studies, S. torvum cv. Hawk was found to be resistant to M. incognita (Chitwood, 1949;Kofoid andWhite, 1919) (both Mi-1.2-virulent andavirulent isolates), M. javanica, M. arenaria, andM. luci (Carneiro et al., 2014) isolates, but susceptible to isolate of M. hapla (Chitwood, 1949) from Turkey (Öçal and Devran, 2019;Öçal et al., 2018). Recently, Murata and Uesugi (2020) reported that S. torvum cvs. Torvum vigor, Tonashimu and Torero were susceptible to three M. hapla populations collected from geographically distant areas in Japan.
In tomato, resistant cultivars are widely used to control RKNs. The Mi-1 gene in tomato confers resistance to M. incognita, M. javanica, and M. arenaria (Milligan et al., 1998;Roberts and Thomason, 1986;Williamson and Hussey, 1996), as well as M. luci (Aydınlı and Mennan, 2019). However, Mi-resistancebreaking M. incognita and M. javanica populations were reported in United States of America, Spain, Greece, Turkey, and Israel (Devran and Söğüt, 2010;Iberkleid et al., 2014;Kaloshian et al., 1996;Ornat et al., 2001;Tzortzakakis et al., 2005). In addition, a virulent population of M. luci has been reported from Turkey (Aydınlı and Mennan, 2019). Meloidogyne luci poses a phytosanitary risk as the species was included on the EPPO Alert List of harmful organisms in (EPPO, 2017. In previous studies, S. torvum cultivars Tonashimu, Torero, Torvum vigor, Hawk, Brutus, Espina, Salutamu, and Torpedo were found to be resistant to Mi-1.2-virulent isolates of M. incognita from Japan and Turkey and Mi-1.2virulent M. javanica from Spain (Garcia-Mendivil et al., 2019). Thus, knowledge of the response of S. torvum rootstock cultivars to various RKN populations is important for control of both Mi-1.2-avirulent and virulent populations of root-knot nematodes, which are likely to be present in mixed populations in vegetable growing areas of Turkey. In Turkey, two S. torvum rootstocks, Hawk and Boğaçare used to control RKNs in eggplant growing areas. Solanum torvum cv. Hawk has been described as resistant to some RKN isolates from Turkey (Öçal et al., 2018). As far as the authors are aware, there is no information about the response of commercial S. torvum cultivars to Mi-1.2 virulent isolates of M. javanica and M. luci from Turkey or the response of S. torvum cv. Boğaçto root-knot nematodes isolates. Here, we report the degree of resistance of S. torvum cultivars Hawk and Boğaç to avirulent and virulent isolates of M. incognita, M. javanica, and M. luci from Turkey.

Plant material
The two S. torvum rootstocks Hawk and Boğaç and a commercial eggplant cultivar Faselis F 1 were used in this study (Table 1). All seeds were purchased from companies. Seeds were sown in vials containing vermiculite, perlite, and peat (v:v:v, 1:1:1) and incubated in controlled environment at 25 ± 5°C. Seedlings with two true leaves were transplanted singly to 250 ml pots containing sterilized sandy soil (75% sand, 15% silt, and 10% clay) after germination. The plants were watered when needed.

Root-knot nematode culture
Four M. incognita, four M. javanica, and two M. luci isolates were used in the experiment (  (Aydınlı and Mennan, 2019;Devran and Söğüt, 2010). Each isolate was raised from single egg mass to obtain pure cultures. The plants were removed 60 days after root-knot nematode inoculation, and their roots were washed under water. Egg masses were picked from plant roots using a needle and incubated in a modified Baermann funnel technique (Hooper, 1986) at 25°C room temperature for 1 day. Then, the J2s hatched from the egg masses were collected and counted under a light microscope according to the method described in previous studies (Özalp and Devran, 2018;Öçal et al., 2018).

Nematode inoculation
Eggplant seedlings with three true leaves were inoculated with 1,000 one-day-old J2s. The J2s were dropped using a Pasteur pipette into a 2-cm deep hole around the stem base according to the method described in previous studies (Öçal and Devran, 2019;Öçal et al., 2018). Each nematode speciesrootstock combination was replicated five times in a completely randomized design and the experiment was repeated twice under same conditions. The pots were maintained in a growth chamber at 24 ± 1°C with a 16:8 hr (light:dark) photoperiod and 65% relative humidity. Sixty days after inoculation, the plants were removed, and their roots were washed under water.
Each root system was dipped in a 0.15 g/L phloxine B solution for 10 min to identify egg masses, according to Öçal et al. (2018).

Statistical analyses
Data on number of egg masses were analyzed using the SAS statistical software (SAS Institute, Cary, NC). Data were log transformed [log10(x + 1)] to homogenize the variances, and then subjected to analysis of variance (ANOVA). Separation of treatment means was performed using Tukey's HSD tests (p < 0.05).  (Table 3). Differences among the number of egg masses produced by RKN isolates on roots of seedlings were not statistically significant for S. torvum rootstocks Hawk and Boğaç, but the numbers on these rootstocks were significantly different from those on

Discussion
Eggplant is one of the most important vegetable crops for human nutrition. However, many pathogens and pests negatively affect vegetable production. Root-knot nematodes (RKNs) are one of the most important pathogens causing yield losses in vegetable growing areas. They inhibit the plant's water and nutrient intake from soil and cause galls on its roots. Different management methods are used to control RKNs and use of resistant plants is one of the most important (Boerma and Hussey, 1992). Previous studies showed that S. melongena cultivars were susceptible to M. incognita, M. javanica, and M. arenaria (Daunay and Dalmasso, 1985;Devi and Sumita, 2015;Nayak and Pandey, 2015;Öçal and Devran, 2019;Ullah et al., 2011 (Dhivya et al., 2014;Öçal and Devran, 2019;Öçal et al., 2018;Tzortzakakis et al., 2006;Uehara et al., 2017) but susceptible to M. hapla (Murata and Uesugi, 2020;Öçal et al., 2018). Other studies also reported that S. torvum cultivars were resistant to Mi-1.2 virulent M. incognita isolates from Japan (Uehara et al.,  (Carneiro et al., 2014), Guatemala (Janssen et al., 2016), Portugal (Maleita et al., 2017), and Turkey (Aydınlı and Mennan, 2019), and so sources of resistance to virulent isolates of this RKN are of importance to growers in these countries. Applications of chemical control against plant parasitic nematodes are expensive and have been banned due to their adverse effects on the environment and harmful residual effects on human health. Therefore, resistant varieties and grafted seedlings are preferred due to their lower cost, their effectiveness, and their long-term and environmentally friendly protection compared to chemicals (Barrett et al., 2012;Cook and Starr, 2006;Lopes et al., 2019;Williamson and Kumar, 2006). Also, Mi-1.2 virulent RKNs are becoming widespread in vegetablegrowing areas. Therefore, new sources of resistance are essential for planting in fields infested with RKNs. The results of this study show that S. torvum is a non-host for M. incognita, M. javanica, and M. luci isolates from Turkey and is, therefore, a valuable tool for controlling M. incognita, M. javanica, and M. luci populations irrespective of their (a)virulence status to the Mi-1.2 gene. Further studies are needed to test other S. torvum rootstock varieties to determine how their yields compare with those of susceptible commercial cultivars.