Leaf mineral composition of grafted eggplant grown in soil infested with Verticillium and root‑knot nematodes

– The objective of this work was to determine differences in leaf mineral composition between ungrafted and grafted onto ( Solanum torvum ) eggplant ( Solanum melongena ), cultivars 'Faselis' and 'Pala', grown in a soil infested with Verticillium dahliae and Meloidogyne incognita , or in a noninfested soil. Grafting increased leaf P and Mn concentrations, and decreased N concentrations, in both soils. Grafting also enhanced leaf Ca concentration of 'Pala', but it did not affect that of 'Faselis' depending on the cropping year. Leaf Mg concentration of grafted plants in infested soil was lower than that of ungrafted ones in noninfested soil. Results showed that, under the same fertilization program, the grafted 'Faselis' plants used the nutrients more efficiently than the 'Pala' ones. Use of S. torvum as a rootstock for 'Faselis' resulted in an effective protection against multiple pathogen infestation. Fertilization may be necessary when grafted 'Faselis' plants are grown in a soil infested with the pathogens, since grafting and infestation generally decrease leaf N, Mg, Ca and Fe concentrations, either by reducing the nutrient concentrations directly or by increasing leaf Mn concentration.


Introduction
Eggplant (Solanum melongena L.) is susceptible to numerous diseases and parasites, particularly bacterial ones, Fusarium and Verticillium wilts, nematodes and insects (Collonnier et al., 2001).Soil-borne pathogens and pests such as Verticillium, Fusarium and Meloidogyne spp.may cause up to 78% yield losses (Bletsos et al., 2003).Difficulties in chemical control of these diseases, absence of crop rotation, and sexual barriers between eggplant and its wild relatives that hinder breeding indicate that the only short term practical solution for the problem is to graft susceptible eggplant cultivars onto rootstocks with multiple pathogen resistance (Bletsos et al., 2003).Plant production by grafting has become a common practice to control soil-borne pathogens, especially for some Cucurbitaceae and Solanaceae species in greenhouses S. Çürük et al. Pesq. agropec. bras., Brasília, v.45, n.8, p.879-885, ago. 2010 in Japan, Korea, China and in some Asian and European countries (Lee, 1994;King et al., 2008).
Eggplant is a popular vegetable in Turkey, and protected cultivation accounts for 20% of the total production.Grafting is a popular technique in protected cultivation of watermelon and melon in Turkey.However, although commercial growing of eggplant is constrained due to soil-borne pathogens such as Verticillium dahliae (Altınok & Kamberoğlu, 2005) and Meloidogyne incognita (root-knot nematode) (Elekçioğlu et al., 1994), grafting in eggplant is not a common practice even in protected cultivation.Grafting of eggplant on rootstocks with multiple pathogen resistance may be used intensively in protected cultivation in the near future.
The most common rootstocks for eggplant are tomato (Ioannou, 2001) and related wild species such as Solanum torvum Sw., Solanum integrifolium Poir.and Solanum sisymbriifolium Lam.(Lee, 1994;Bletsos et al., 2003).Solanum torvum was reported to be resistant to Verticillium and bacterial wilts, root-knot nematode, and mycoplasma (Collonnier et al., 2001;Kashyap et al., 2003).Bletsos et al. (2003) have found that grafting of eggplant onto S. torvum and S. sisymbriifolium had positive effects on plant growth, yield and resistance to the incidence of V. dahliae, without changing fruit quality.The method of disease control provided by grafting is not well understood.It is presumed that the primary method is by avoidance, when the rootstock is resistant to the pathogen.This has been confirmed by observations in which suckers or other intact roots from the scion confer susceptibility to the grafted plant (King et al., 2008).Another mechanism is the allelopathic suppression exhibited by the root exudates from the rootstock.Root exudates from tomato rootstocks of eggplants were shown to directly inhibit the spore germination and mycelium growth of Verticillium wilt, while the root exudates of ungrafted eggplants enhanced the mycelium growth (Liu et al., 2009).
Grafting may influence plant vigor expressed through variations in growth, yield and product quality.The mechanisms that determine these effects can be diverse and can be attributed to the synthesis and translocation of plant growth regulators, to the size of the root system, and to water and nutrient uptake and assimilation (Lee, 1994;Leonardi & Giuffrida, 2006).Available information on grafted plants nutrition is not exhaustive, and in many cases is limited to cucurbits (Ruiz et al., 1997;Pulgar et al., 2000;Colla et al., 2006;Leonardi & Giuffrida, 2006).
Pathogens alter the nutrition of the plant in diverse ways.Some pathogens may immobilize nutrients in the rhizosphere or in the infected tissues; others impair the translocation and utilization of nutrients directly by malfunctioning of the vascular system, while still others cause deficiency or hyper accumulation and nutrient toxicity by changing membrane permeability, or mobilization toward infection sites (Huber, 1978;Huber & Graham, 1999).Sometimes, different pathogens infect the crops simultaneously.Several reports implicate nematode root-knot lesions in breaking down the resistance of tomato, eggplant, cotton and peppermint plants to Fusarium or Verticillium wilts, due to the increase in disease severity (Hasan & Khan, 1985;Katsantonis et al., 2005).Katsantonis et al. (2005) reported that root-knot nematode infestation in cotton could cause a decrease in total protein and carbohydrate levels and an increase in peroxidase activity in xylem fluids.These could be the mechanisms by which infection by Fusarium or Verticillium wilts is enhanced by root-knot nematode invasion.There are few reports concerning the effects of different rootstocks on mineral uptake and nutrition in tomato and eggplant (Suzuki & Morishita, 2002;Leonardi & Giuffrida, 2006;Arao et al., 2008), and there are no reports on the effect of S. torvum rootstock on leaf mineral composition when soil is infested with multiple pathogens.
The objective of this study was to investigate the effect of grafting eggplant onto S. torvum on leaf mineral composition of eggplant cultivars 'Faselis' and 'Pala' grown in noninfested or infested soils with V. dahliae Kleb.and M. incognita.
The experimental design consisted of a factorial randomized complete block in a split plot arrangement, with three replicates per treatment.The factor pathogen (noninfested or infested soil with V. dahliae and M. incognita) was assigned to the main plots of each block; and cultivar ('Pala' or 'Faselis' F 1 ) and grafting (grafted on S. torvum or nongrafted) factorial combinations were randomly assigned to the subplots of each main plot.Based on soil analysis, the total amount of applied N-P 2 O 5 -K 2 O was 980-260-1,560 kg ha -1 (Savvas et al., 2008).All the manure and the fertilizer applied before planting were immediately incorporated into the soil.The fertilizer applied after planting was dissolved in water before using.Grafting by the budding method was used to produce grafted eggplant plantlets (Çürük et al., 2009).Seeds of cultivars 'Faselis' F 1 (De Ruiter Seeds, Bergschenhoek Holland) and 'Pala' (Atatürk Central Research Institute, Yalova-Bursa, Turkey) were sown in a sterile substrate, composed of 50% peat and perlite, in August of 2003 and 2004.Ungrafted control and grafted transplants with 5-6 leaves were transplanted to plastic bags containing 25 L of above mentioned soil mixture.Transplanting was on 15 th and 22 nd October in 2003 and 2004, respectively, at a density of 2.66 plants m -2 (0.75x0.5 m).Grafted and control seedlings had similar height and leaf number at this stage.Unless otherwise noted, measurements were made on five plants per replicate.
Verticillium dahliae EgVd3 isolates and egg masses of M. incognita were obtained from diseased eggplants and inoculated in the soil about two and four weeks after transplanting, respectively (Çürük et al., 2009).For Verticilium inoculation, conidial suspension was adjusted to a concentration of 10 6 conidia mL -1 with a hemacytometer.The conidial suspension (250 mL) was added to the substrate around each seedling using a micropipette.Each plant used in the infested plot was infected with 1,200 nematode eggs placed into a 5-cm depth hole near the roots.Leaf symptoms and disease indices were calculated as reported by Çürük et al. (2009).
Plant analyses for nitrogen, phosphorus, potassium, calcium, magnesium, iron, manganese, zinc and copper were carried out to compare the nutritional status of eggplant plants grown under the different treatments.In both cropping years, leaf samples (most-recently-matured whole leaf plus petiole) were collected approximately six months after transplanting, and dried at 65 o C for 72 hours.After drying, the samples were ground to 20 mesh sieve size.Tissue powder was ashed at 550 o C for about eight hours and dissolved in 3.3% HCl.The concentrations of K, Ca, Mg, Fe, Mn, Zn and Cu were assessed through atomic absorption spectrophotometry (Jones Junior, 1972; Analytical methods for atomic absorption spectrophotometry, 1976).Nitrogen and P concentrations were determined by the Kjeldahl and Barton methods, respectively (Jones Junior et al., 1991).
Statistical analyzes were done using SAS (SAS Institute, 1999) and MSTAT-C (Freed & Eisensmith, 1989).When year or interactions involving year were not significant, data of the two cropping years were combined and analyzed by a general linear model (GLM) ANOVA, and means were shown.Otherwise, data of each year were analyzed and shown separately.Duncan's multiple range test was used for pairwise comparisons.

Results and Discussion
The leaf N, P, Mg and Cu concentrations were not significantly affected by year or interactions involving year.Therefore, the average data of the two years were presented for these variables ( N concentration from 48.2 to 45.0 g kg -1 (6.6%).Grafted plants generally have bigger shoots than ungrafted ones, especially in the 'Pala' cultivar (Çürük et al., 2009).This may have caused a dilution effect, which would explain the lower N concentrations in grafted eggplants.It was reported that leaf nitrogen concentration decreased in watermelon grafted on pumpkin squash (Pulgar et al., 2000).However, it has been found that grafting increased leaf N concentration in eggplant (Leonardi & Giuffrida, 2006), tomato (Fernandez-Garcia et al., 2004) and cucumber (Rouphael et al., 2008) grafted onto tomato or interspecific hybrid rootstocks, tomato rootstock, and Cucurbita maxima x C. moschata hybrid, respectively.According to Ruiz et al. (1997), the effect of grafting on leaf N concentration in melon differed depending on the rootstocks (C.maxima x C. moschata hybrids) used.
The effects of grafting, cultivar, pathogen-grafting and pathogen-cultivar interaction on leaf Mg concentration were significant.By grafting, leaf Mg concentration decreased by 53.6% (from 7.89 to 3.66 g kg -1 ) in noninfested soil, but increased by 42.3% (from 4.49 to 6.39 g kg -1 ) in infested soil.2004) suggested that leaf Mg concentration of tomato grafted onto tomato rootstock was either higher or similar to the ungrafted ones.In the present study, infestation reduced leaf Mg concentration (from 5.61 to 4.24 g kg -1 ) by 24.4% in 'Pala', but in 'Faselis' the difference between leaf Mg concentration in infested (6.64 g kg -1 ) and noninfested (5.94 g kg -1 ) soils was not significant.
Leaf Cu concentration was not affected by pathogen, grafting, cultivar or interactions of these factors (Table 1).It was reported that there was no significant difference between leaf Cu concentration of grafted and ungrafted plants of cucumber grown on regular Cu concentration in the nutrient film technique (Rouphael et al., 2008).
Concerning leaf Ca concentration, grafting-cultivar interaction in both cropping years, and pathogen, cultivar and pathogen-grafting interaction in 2004/2005 were significant.In 2003/2004, grafting increased leaf Ca concentration (from 21.1 to 30.3 g kg -1 ) significantly by 43.6%only in 'Pala'.In 2004/2005, grafting enhanced leaf Ca concentration in 'Pala' by 22.2% (from 38.3 to 46.8 g kg -1 ), but reduced it in 'Faselis' by 15.3% (from 64.9 to 55.0 g kg -1 ) .By infestation, leaf Ca concentration decreased by 28.8% in grafted plants (from 59.4 to 42.3 g kg -1 ), but the decrement in leaf Ca concentration in ungrafted ones (from 54.1 to 49.1 g kg -1 ) was not significant.It was reported that leaf Ca concentration increased in tomato by grafting onto tomato rootstock (Fernandez-Garcia et al., 2004).Arao et al. (2008) suggested that leaf-stem Ca concentrations of self-rooted eggplant cultivars were either higher than or similar to S. torvum in Cd-polluted soil.Ruiz et al. (1997) indicated that Ca concentration in grafted melon plants was not significantly different than that of ungrafted ones.In the present study, the internal fruit rot, as reported by Savvas et al. (2008), was infrequently observed in 'Faselis' relating to pathogen inoculation and grafting.Internal fruit rot is most likely to be a calcium-related physiological disorder caused by low Ca translocation to the fruit (Savvas et al., 2008).
Grafting in both cropping years significantly affected leaf Mn concentration.Leaf Mn concentrations of ungrafted plants in 2003/2004 and 2004/2005 (70.6 and 73.5 mg kg -1 , respectively) were 46.9 and 67.5% lower than those of grafted ones (133.0 and 225.9 mg kg -1 ).This finding is not consistent with the results by Rouphael et al. (2008), which showed that grafting did not affect leaf Mn concentration in cucumber.Arao et al. (2008) have reported that leaf-stem concentrations of self-rooted eggplant cultivars were similar to S. torvum.Leaf Mn concentration of 'Faselis' (106.4 mg kg -1 ) was 9.5% higher than that of 'Pala' (97.2 mg kg -1 ) in 2003/2004, but the difference between the cultivars was not significant in 2004/2005.) Ca (g kg - 1) Mn (mg kg -1 ) Fe (mg kg -1 ) Zn (mg kg -1 ) K (g kg - 1) Ca (g kg - 1) Mn (mg kg Means followed by equal letters, in the columns, do not differ by Duncan's multiple range test, at 5% probability. S. Çürük et al. Pesq. agropec. bras., Brasília, v.45, n.8, p.879-885, ago. 2010 Leaf Fe concentration was affected by grafting, cultivar, grafting-cultivar and pathogen-cultivar interactions in 2003/2004, and by grafting, pathogen-grafting and pathogen-grafting-cultivar interactions in 2004/2005. In 2003/2004, leaf Fe concentration of 'Pala' diminished 31.4% (from 146.0 to 100.2 mg kg -1 ) by grafting and increased 35.6% (from 104.5 to 141.7 mg kg -1 ) by infestation.In 2004/2005, leaf Fe concentration was enhanced 22.8% by infestation in ungrafted 'Faselis' plants, but it decreased 21.2% in grafted ones (Table 2).Iron deficiency symptoms that are induced by excess of Mn concentration, due to grafting, as reported by Savvas et al. (2008), were also observed in 'Faselis' during the winter periods of the experiments.Similar symptoms were also observed in S. torvum and were mild in the summer.Arao et al. (2008) reported that the differences in leaf-stem Fe concentrations, between self-rooted plants of S. melongena and S. torvum, were not significant, and that there were no significant differences between leaf-stem Fe concentrations of eggplants grafted on S. integrifolium and S. torvum.A similar result was reported by Rouphael et al. (2008).
Concerning leaf Zn concentration, grafting-cultivar interaction in 2003/2004, and pathogen, grafting, grafting-cultivar and pathogen-grafting-cultivar interactions in 2004/2005 were significant.In 2003/2004, grafting reduced leaf Zn concentration by 18.4% (from 20.1 to 16.4 mg kg -1 ) in 'Pala', but with no significant differences in Zn concentration between grafted and ungrafted 'Faselis' plants.In 2004/2005, leaf Zn concentration in ungrafted 'Pala' and grafted 'Faselis' plants decreased 35.9% and 18.3%, respectively, by infestation (Table 2).Arao et al. (2008) reported that leaf-stem Zn concentration of self-rooted S. torvum was higher than that of S. melongena, while there was no significant difference in leaf-stem Zn concentration in eggplant between grafted on S. torvum and on S. integrifolium.Rouphael et al. (2008) showed that grafting did not significantly affect Zn concentration of cucumber leaf.
In the present study, leaf analyses for N, P, K, Ca, Mg, Fe, Zn, Mn and Cu showed that the eggplants in the experiments were adequately fed throughout the growth period.The ranges of nutrient concentrations recorded in control plants (ungrafted ones in noninfested soil) were within the order of "sufficient" for N, P, Mg, Cu, Mn, Fe and Zn in both cropping years and for K in 2003/2004, while they were higher than "sufficient" for Ca in both years and for K in 2004/2005(Warr, 1993)).
Vegetative growth and fruit yield were affected by cultivar characteristics according to Çürük et al. (2009).This could be attributed to the different growth characteristics, as 'Pala' is more vigorous and produces less fruit than 'Faselis'.This could also be attributed to the different graft affinity and compatibility with the rootstock.Solanum torvum is a vigorous rootstock, and graft combination of a vigorous cultivar with an equally vigorous rootstock reduced the amount of fertilizer required for the same yield (Suzuki & Morishita, 2002).However, combination of a vigorous rootstock with a weak cultivar (Faselis) is more profitable than that of a vigorous rootstock with a vigorous cultivar (Pala) (Çürük et al., 2009).'Faselis' also produced higher fruit yield than 'Pala' (Çürük et al., 2009).This showed that the grafted plants of 'Faselis' used the nutrients more efficiently than the 'Pala' ones.However, 'Faselis' may need additional nutrients, especially N, Mg, Ca and Fe during the winter, since the pathogens and grafting decrease the nutrients, either by reducing nutrient concentrations directly or by increasing leaf Mn concentration.

Conclusions
1. Grafting decreases leaf N concentration, regardless of soil infestation, and decreases Mg concentration in infested soil.
2. Grafting increases leaf P and Mn concentrations, regardless of soil infestation, and enhances Ca in 'Pala'.
3. Nutrients are more efficiently used by grafted 'Faselis' plants in comparison to 'Pala'.
4. 'Faselis' may need additional N, Mg, Ca and Fe fertilization when grafted onto Solanum torvum and grown in a soil infested with pathogens.
0°C, respectively, in the cropping periods of both years.The mean relative humidity was ca.69 and 67% during the 2003/2004 and 2004/2005 growing periods, respectively.Plants were grown in a greenhouse with steel structure, with 12.8 m width span, 2.5 m gutter height, 4.5 m ridge height, continuous roof

Table 1 )
. Leaf N concentration was affected by grafting, but not by pathogen or cultivar.Grafting decreased leaf S.Çürük et al.

Table 1 .
Leaf macro-and micronutrient average concentrations of the two cropping years 'Faselis' and 'Pala' of eggplant cultivars grown with or without rootstocks, in noninfested or infested (Vericillium dahliae and Meloidogyne incognita) soil.