Potential of wild Solanum stramonifolium accesses as rootstock resistant to soilborne pathogens in tomato crops

Resistant rootstocks is one of the most effective method to control soilborne pathogens in tomato crops. Thus, this study was installed to evaluate the reaction of Solanum stramonifolium accesses to Fusarium oxysporum f. sp. lycopersici (Fol) races 2 and 3 and to Meloidogyne enterolobii (Me). The seedlings were grown in trays and inoculated separately with Fol races 2 and 3 at 50 days after planting by immersing the roots in the spore suspension (1×106 microconidia mL-1). Then, seedlings were transplanted in pots containing sterilized soil and kept in greenhouse conditions. To study the reaction of S. stramonifolium accesses to nematodes, we used 27-day old seedlings that were also planted in pots and inoculated with 6,000 eggs and second-stage juveniles in greenhouse conditions. The experiments were evaluated in the 34th day (Fol) and in the 64th day (Me) after inoculation. The experiment consisted of a randomized block design with five replications, where each plot consisted of one pot with three plants (Fol) and one pot with one plant (Me). We observed that the plants used as controls, susceptible to Fol races 2 and 3 and Me, presented 100% of incidence. All accesses were resistant to Fol race 2 and the accesses CNPH-19, CNPH-22, CNPH-23, CNPH-25, CNPH120, CNPH-122 and CNPH-349 presented multiple resistance to pathogens, indicating great potential for using as resistant rootstock. The CNPH-24, CNPH-119, CNPH-121 and CNPH-336 accesses also presented resistance to nematode. However, they presented slight browning symptoms of vascular tissues when they were inoculated with Fol race 3. This symptom was also observed in the CNPH21, CNPH-107 and CNPH-117 accesses. All other accesses were resistance to Fol race 3 and susceptible to Me.

In Brazil, the most common Meloidogyne species in tomato crops are M. javanica, M. incognita (races 1, 2, 3 and 4) and M. arenaria.However, in 2001, Carneiro et al. (2001) reported large losses in tomato crops in São Paulo State due to the attack of M. enterolobii (syn.M. mayaguensis).Recently, Pinheiro et al. (2015) reported the occurrence of this species in Central Region of Brazil, which made unfeasible the tomato cultivation in such areas.
Currently, three physiological races of F. oxysporum f. sp.lycopersici are known in Brazil, which are able to infect and to cause disease in many tomato host cultivars.Race 3 has been confirmed to be responsible for epidemics outbreaks in tomato crops in the Southeast and Northeast regions of Brazil (Reis et al., 2005;Reis & Boiteux, 2007;Barbosa et al., 2013;Gonçalves et al., 2013), although races 1 and 2 are the most common in tomato producing areas.
The majority of commercial tomato cultivars grown in Brazil have the dominant gene Mi, which confers resistance to the prevailing nematode species in the country.However, the gene Mi do not provide resistance to M. enterolobii in many crops, including tomato (Pinheiro et al., 2011).According to Trudgill (1991), little is known about sources of resistance to nematodes in vegetables, such as Solanum species.The majority of tomato cultivars and rootstocks traditionally grown in Brazil are resistant to races 1 and 2 of F. oxysporum f. sp.lycopersici.However, resistant cultivars and rootstocks to race 3 are scarce, Race 3 is disseminated to the main production areas of tomato in Brazil (Reis & Boiteux, 2007;Gonçalves et al., 2013).
Therefore, the use of resistant tomato rootstocks can be an alternative method to be used in the control of soilborne pathogens responsible for causing diseases in tomato crops (Lopes & Mendonça, 2016).In this sense, plant species of the genus Solanum, sub-genre Leptostemonum have been evaluated in order to use them as rootstock for tomato.Mendonça et al. (2005) evaluated the performance of 'Santa Clara' tomato grafted onto S. lycocarpum rootstock on soil infested with Ralstonia solanacearum and obtained higher yields in comparison to the non-grafted.These authors also reported 95% of compatibility between S. lycocarpum accesses and 'Santa Clara' tomato.Mattos et al. (2011) evaluated the reaction of S. stramonifolium and S. asperolanatum and reported resistance of accesses to M. incognita race 1.In addition, they found that accesses of S. stramonifolium, S. paniculatum and S. subinerme showed resistance to M. enterolobii.Pinheiro et al. (2011) evaluated the reaction of the same accesses of S. stramonifolium, S. paniculatum and S. subinerme evaluated by Mattos et al. (2011) to root-knot nematode and they found that these rootstocks were also resistant to M. incognita race 1 and M. javanica.
Considering the encouraging results reported up to date to wild Solanum resistant rootstocks against the main soil pathogens in tomato crops, we developed a study to evaluate the reaction of S. stramonifolium accesses to the fungus F. oxysporum f. sp.lycopersici races 2 and 3 and to the nematode M. enterolobii.

MATERIAL AND METHODS
The assays were conducted in the Laboratory of Plant Pathology and Nematology and in a greenhouse located at Embrapa Hortaliças, Brasília-DF, Brazil, during the period of September to December 2014.
Tw e n t y -t w o a c c e s s e s o f S .stramonifolium were evaluated separately regarding the reaction to F. oxysporum f. sp.lycopersici race 2 and 3 and to M. enterolobii,  In the experiments of F. oxysporum f. sp.lycopersici race 2 and 3 the tomato cv.Santa Clara was used as susceptible control to pathogen, while in the reaction experiments to nematodes, we used tomato plants cv.Nemadoro and cv.Rutgers, which are resistant and susceptible control to pathogen, respectively.
For inoculum preparation, we used three discs (5 mm diameter) removed of pure pathogen colonies.Then, the discs were transferred to Erlenmeyer flasks containing 250 mL of culture medium made of potato dextrose broth (BD) and maintained at temperature of 23 to 27°C under constant agitation (90 rpm).After seven days of incubation, the liquid culture medium containing the fungus microconidia was filtered on sterile gauze, and its concentration was measured by hemocytometer, according to Santos (1997).In order to identify races and accesses in all reaction experiments, we used the same concentration of the inoculum suspension (1×10 6 microconidia mL -1 ).

Meloidogyne enterolobii
The nematode species were obtained from tomato plants presenting gall symptoms on roots.The identification process was done by exam of the perineal cuts of adult females extracted from galls and confirmed by standard isoenzymes (Carneiro & Almeida, 2001).
Eggs and any second stage juveniles (J2) were collected of M. enterolobii females previously obtained and inoculated in tomato cv.Rutgers plants for multiplication of inoculum.These plants were grown in 3.0 L pots containing sterilized substrate and maintained in greenhouse.Fifty days after inoculation, eggs and J2 were extracted from tomato roots [Hussey & Barker (1973) modified by Bonetti & Ferraz (1981)] and quantified under microscope stereoscope.For the experiment, the inoculum suspension was adjusted to 6,000 eggs and J2 per plant, and distributed in 5 mL of suspension.

Experiments conduction
All tomato and S. stramonifolium accesses were planted in 72-cell trays containing substrate, which was composed of vermiculite and carbonized pine bark.Seedlings were daily irrigated according to necessity and kept in greenhouse throughout the experimental period.

Fusarium oxysporum f. sp. lycopersici races 2 and 3
Seedlings of S. stramonifolium accesses and tomato plants were inoculated separately with the races of the pathogen after 50 days in case of S. stramonifolium and 25 days for tomatoes (Santos, 1997).After that, seedlings were removed from the trays, and roots washed in tap water in order to remove the substrate.Roots were cut about 4 cm from the stalk by using a sterile scissor.Then, roots were completely immersed (2 min.) in the inoculum suspension (1 × 10 6 microconidia mL -1 ) and transplanted to 1.5 L pots, filled with autoclaved substrate, which was a mixture of 85% of sifted "cerrado" underground, 5% of dry rice husk and 10% carbonized rice husk (v:v).The substrate was enriched with 100 g of dolomite lime, 200 g of superphosphate and 60 g of ammonium sulfate.
The experiment consisted of randomized block design with five replications where each plot consisted of one pot containing three plants.

Meloidogyne enterolobii
The 27-day old seedlings of S. stramonifolium accesses and tomato plants used as controls were also transplanted into pots (4.5 L) filled with same substrate described in the previous experiment.
The accesses of S. stramonifolium a n d c o n t r o l s w e r e i n o c u l a t e d , distributing 5 mL of the suspension (6,000 eggs and J2) per plant, around the neck of the seedlings, with 2 cm range and depth approximately.
The experiment consisted in a randomized block design with five replications, where each plot consisted of one pot with one plant.

Experimental assessments
Fusarium oxysporum f. sp.lycopersici races 2 and 3 The disease symptoms were evaluated 50 days after inoculation, based on a scale of notes, wherein: 0= no symptoms, 1= plants without wilting or yellowing symptoms but with vascular browning, 2= plants with intense vascular browning and beginning to wilt or with yellow leaves, 3= plants with intense wilting associated with yellowing and leaf drop, 4= dead plants (Aguiar et al., 2013).
Based on the notes, we were able to determine the disease index (DI), that was calculated by using the formula DI (%) = 100.Σ [(fv) / (nx)], where f= is the number of plants with same note, v= observed note, n= total number of evaluated plants and x= maximum rating scale plants (McKinney, 1923).It is important to highlight that only the genotypes with note zero were considered as resistant.
Immediately after evaluation, the plants with symptoms of the pathogen were identified and sent to the laboratory for isolation of the fungus in potatodextrose-agar (PDA) culture medium.Conidia suspensions of each isolate were prepared from pure cultures of the pathogen, which were inoculated on susceptible tomato plants cv.Santa Clara.Then, the pathogenicity of these isolates was confirmed in all S. stramonifolium plants, which present darkened vascular bundles symptoms.

Meloidogyne enterolobii
The evaluation of M. enterolobii was performed 64 days after inoculation, where plants were removed from the pots and identified.Roots were washed thoroughly in tap water and processed by using the technique developed by Hussey & Barker (1973) and modified by Bonetti & Ferraz (1981).Then, the final population of eggs and J2 obtained from each root system was quantified under microscope stereoscope.
The reproduction factor (RF) was obtained by the ratio between the final densities (Pf) and initial (Pi) of the nematodes, according to the formula: RF = Pf / Pi (Oostenbrink, 1966).Pi was considered the inoculum distributed at the time of inoculation, where, 6,000 eggs and J2 per pot.Pants with RF= 0 were considered as immune, those resistant to RF<1.0 and those susceptible to RF>1.0.
The reproduction factor data (RF) were transformed to √(x+1), submitted to the analysis of variance in statistical software Sisvar ® (v.4.5).Averages were grouped by the Scott-Knott test (p≤0.05).

RESULTS AND DISCUSSION
All 22 accesses of S. stramonifolium showed resistance to F. oxysporum f. sp.lycopersici race 2 (Table 1), without expressing any symptoms of the disease.However, seven accesses of S. stramonifolium  presented mild symptoms of vascular browning when inoculated with race 3 of the pathogen.These accesses presented disease index (DI) of 3.10, 6.30, 6.30, 9.40, 12.50, 15.60 and 18.80, respectively, and they were considered susceptible.The tomato cv.Santa Clara, used as susceptible control for both pathogen races, showed the symptom of disease in 100% of the plants and presented DI= 82.30% to race 2 and 71.90% to race 3.
Regarding the resistance of S. stramonifolium to the nematode M. enterolobii, we observed that there was great variability among the accesses (Table 1).Eleven accesses (50.0%)These results highlight the potential use of accesses of S. stramonifolium as resistant rootstocks against F. oxysporum f. sp.lycopersici races 2 and 3 in tomato crops.Similar results were found by Pinheiro et al. (2011), where the selected accesses of S. stramonifolium and other wild Solanum species were resistant to M. incognita race 1 and to M. javanica.In addition, Mendonça et al. (2005)  plants.The compatibility of tomato cv.IPA-6 (Farias et al., 2013) and cv.Santa Adélia (Simões et al., 2014) grafted onto S. stramonifolium and S. lycocarpum rootstocks was evaluated and higher yield and graft compatibility with tomato cultivars was obtained.
Therefore, according to literature some accesses of wild Solanum species presented resistance to multiple soil pathogens, not interfering negatively on the tomato production.In the present work, we could find similar results, where seven accesses of S. stramonifolium showed multiple resistance,  indicating that these accesses possess high potential to be used as resistant rootstocks to diseases in tomato grown in infested areas.According to Farias et al. (2013) it is important to keep focusing on studies such as the present one, in order to select new tomato cultivars, compatible with wild Solanaceae rootstocks, which will help to control soil pathogens and increase tomato productivity.However, the knowledge about the genes involved in the resistance reactions and in the defense mechanisms involved in the interactions between accessions of S. stramonifolium and soil pathogens need to be elucidated.
showed complete resistance to the nematode, with reproductive factors (RF) lesser Potential of wild Solanum stramonifolium accesses as rootstock resistant to soilborne pathogens in tomato crops