Meloidogyne incognita resistant strains of leaf lettuce

1 Department of Agronomy, Universidade Estadual Paulista‟Júlio de Mesquita Filho‟, 14884-900, Jaboticabal SP, Brazil. 2 Department of Agronomy, Universidade Federal Rural de Pernambuco, 52171-900, Recife, Pernambuco, Brazil. 3 Instituto Agronômico de Pernambuco, 55612-010, Vitória de Santo Antão, PE, Brazil. 4 Department of Plant Science, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil.


INTRODUCTION
Lettuce is a vegetable often affected by nematode galls induced by the root-knot nematode (Meloidogyne spp.).Galls form on roots of infected plants, and consequently block the plant"s uptake of water and nutrients from the soil.This prevents normal development of the lettuce head and results in reduced commercial value (Charchar and Moita, 1996).Nematodes are sometimes chemically controlled; however, these products are toxic and often have a residual effect.Therefore, growing nematode resistant cultivars is a more effective form of control.
Inheritance studies done with crossings of the cultivars Regina-71 (susceptible, flat-leaved) and Grand rapids (resistant, curly-leaved) showed that resistance for both M. incognita (Gomes et al., 2000) and M. javanica (Maluf et al., 2002) is controlled by a single genetic locus.Carvalho Filho et al. (2011a) also found that resistance to the M. incognita race 1 in the cultivars Grand Rapids and Salinas-88 is controlled by alleles located in different loci with independent segregation, and the loci which confers resistance in Salinas-88 was assigned the name Me2.Despite the monogenic inheritance, this characteristic is strongly influenced by the environment and in the case of 'Salinas 88', there are still other modifiers (Carvalho Filho et al., 2008).
The monogenic inheritance for this trait is reinforced by studies involving segregating populations.From the crossing of 'Regina-71' and 'Grand Rapids', 10 F 2:3 lettuce progenies homozygous for resistance were selected (Fiorini et al., 2007).In another study involving , 12 F 4 progenies homozygous for resistance were selected (Carvalho Filho et al., 2007).When comparing 11 advanced lines of lettuce [("Regina-71" × "Grand Rapids") × "Elisa"], six lines were found to be homozygously resistant to M. javanica (Ferreira et al., 2011).These results demonstrate the possibility for selection of resistant homozygous families from crossing contrasting parents.
Studies show that by crossing cultivars with genetic traits of interest, promising offspring can be selected for use as a source of favorable alleles, or perhaps, even be released as a new cultivar (Diamante et al., 2013).Carvalho Filho et al. (2011b) selected heat tolerant progenies when they crossed the Salinas × Grand rapids cultivars.Ferreira et al. (2011), working with advanced lines originating from a cross between Grand rapids and Regina-71, achieved lines resistant to root-knot nematode.
Thus, the objective of this study was to select offspring of butter head-type lettuce crosses resistant to M. incognita spp.for cultivar testing.

MATERIALS AND METHODS
Three experiments were conducted to evaluate the performance of various progenies of advanced lettuce crosses.The first and second experiment were performed at two different times at the Agronomic Institute of Pernambuco -IPA, located in Vitória de Santo Antão, Pernambuco state, Brazil (08° 08" 00"" S, 35° 22" 00"" W; 146 m altitude; 19°C annual temperature).The first was performed from October to November, 2012 (minimum temperature of 20.6°C, and maximum of 36°C), and the second from March to April, 2013 (minimum temperature of 23.3°C and maximum of 34.8°C).The third experiment was conducted in the Greenhouse and Nematology Laboratory of the Plant Pathology Department, Federal Rural University of Pernambuco, Recife, Pernambuco, Brazil (8° 04' 03" S, 34° 55' 00" W; 4 m average altitude; 19°C average temperature) from March to April, 2013.
The lettuce seeds were sown in September, 2012, in 128-celled polystyrene trays filled with a commercial substrate.Two to three seeds were planted in each cell.After development of the first true leaves, the plants were thinned to only one plant per cell.Transplanting occurred thirty days after sowing.Three plots measuring 14 m × 1 m in a randomized block design with three replications were used.Each plot was considered a block, and cultivation practices were performed as recommended for the region (Souza et al., 2008).Fertilization was performed as recommended by Cavalcanti (1998) and 15 days after fertilization, mulching with castor bean pomace was performed.Weeding was done when necessary.
The plants were assessed 30 days after transplanting and the variables analyzed were: plant diameter (distance between the edges of the plant in cm), plant height (measured with a ruler at ground level), number of leaves (leaves that were 3 cm above the basal leaves), fresh plant mass (measured from ground level and after cutting off the noncommercial basal leaves), fresh weight of leaves (in kilograms, obtained after counting the total number of leaves), stem length (in centimeters, obtained after counting the number of leaves), stem diameter (measured with calipers), and according to the methodology proposed by Fiorini et al. (2005), the shape of the leaf blade (scores from 1 = wrinkled, to 5 = flat), form of the leaf edge (scores from 1 = cut edge, to 5 = smooth edge), and leaf color (scores from 1 = dark green, to 3 = light green).
In experiment 2, progenies of experiment 1 that stood out as ideal were selected, and so five F4:5 and six F5:6 offspring, together with the cultivars Regiane and Elisa, were used.The experiment employed a randomized design with three replications.Each plot was considered a block with dimensions of 12 m × 1 m.The same variables previously described were again evaluated.
In experiment 3, 24 progenies were evaluated for resistance to M. incognita spp.; seven F5:6 and 17 F4:5 offspring and the two parental cultivars, Grand rapids (resistant) and Regina-71 (susceptible).The seeding was done in polypropylene trays containing a commercial substrate.Fifteen days after transplanting, inoculation with M. incognita eggs was done at a concentration of 800 eggs cell -1 , corresponding to 20 eggs cm -3 .The inoculation, as well as the extraction of the eggs, was made according to the methodology of Hussey and Barker (1973), as modified by Boneti and Ferraz (1981).A randomized block design was used with three replications of eight plants per plot.
Evaluations were performed 45 days after inoculation, using test plants to verify the efficiency of the inoculum.Number of galls with substrate, number of galls without substrate and egg numbers, was evaluated.For the number of galls with substrate (gall index), a rating scale of 1 to 5 was used and based on the following criteria: 1 = few visible (< 10 galls) and small galls (< 1 mm); 2 = few visible galls, but intermediate in size (1 to 3 mm); 3 = intermediate number of galls visible (10 to 30 galls), with few large galls (> 3 mm); 4 = many visible galls (< 30 galls), predominantly large (> 3 mm), with few coalesced galls; and 5 = large and visible galls (> 30 galls), many already coalesced (Fiorini, 2007).For number of galls without substrate (total number of galls), the roots were washed, avoiding a direct water jet on the roots, and the galls were counted.Finally, the number of eggs was accessed.For this, the egg extractions were performed according to the technique of Hussey and Barker (1973) and modified by Boneti and Ferraz (1981).A Motic ® BA310 Optical System stereoscopic microscope was used for counting the eggs.Analyses were performed using the SAS program (SAS Institute, Inc., 2002).Prior analyses of the data found non-normality and the means were transformed by applying the square root methodology according to Box and Cox (1964).The averages of the Grand rapids (resistant) and Regina-71 (susceptible) cultivars were evaluated, comparing them separately to the averages of each  (Cruz, 2013).

RESULTS AND DISCUSSION
There were significant differences for the traits of plant diameter, shape of leaf blade, shape of leaf edge and leaf color when the F 3:4 and F 3:5 progenies were analyzed (Table 1).For plant diameter, the progenies formed two groups; one formed by the F 3:5 offspring and a second group which included both the F 3:4 offspring and the commercial control strains 62 and 41.The averages of these two groups ranged from 22.05 to 25.83 cm for the first group and from 25.30 to 29.38 cm for the second group.These results qualify the F 3:4 progeny for further testing in order to obtain more productive strains.In addition, values for plant diameters ranging from 10.7 to 16.6 cm have been found in the literature (Souza et al., 2008), so according to the classification of Zárete et al., (2010), all progenies tested were considered commercial, as they had a diameter greater than 20 cm.We highlight the progeny AFX-022B-010-17, with an average diameter of 29.50 cm, outperforming all commercial lines and the commercial controls.
There was no significant difference between the means of the genotypes and the cultivar Elisa, thus, these genotypes were classified as flat-leaf blade.However, there was significant difference among the evaluated commercial controls and the strains 41 and 62, which had wrinkled leaf blades (Table 1).In addition, for the characteristic of leaf color, strains 41 and 62 differed from the controls, as well as the F 3:4 progeny and the two F 3:5 progenies, AFX-018C-002-23-22 and AFX-018C-002-23-15 (Table 1).This is mainly due to the selected goal that was carried out for each line of research (Carvalho Filho et al., 2009).This fact is important because it emphasizes the efficiency of selection for color and shape of the leaf blade.Only four strains; AFX-022B-010-14, AFX-018C-005-42-20, AFX-018C-005-44-28, and AFX-018C-005-44-22, did not have leaf blade form and color similar to the respective cultivar, and thus were considered segregating for these characteristics.Phenotypic segregation was observed when evaluating advanced lines and transgressive segregation towards wrinkling leaf blades (Fiorini et al., 2005;Silva et al., 2008).These results show that for these characteristics, the genetic control is found on more than one gene, and these genotypes can be recovered through backcrossing in later generations.
For the characteristics viz., number of leaves, fresh weight of leaves and weight of the plant, which all have high agronomic importance, the progenies did not differ from commercial cultivars.For the number of leaves, these genotypes obtained approximate values of 28, 31, 24, 28, and 25 leaves head , exceeding strains 41 and 62.So, considering all evaluated characters, the offspring selected for evaluation in experiment 2 were: AFX-18C-002-23-15, AFX-020B-006-13 and AFX-022C-010-17.
There were no significant differences for the traits of plant diameter, plant height, fresh weight of plant, stem length, stem diameter and leaf border shape compared to the commercial controls and among the progeny (Table 2).The progenies obtained higher means when compared to the commercial control (Diamante et al., 2013).Similar behavior was observed in both studies, and therefore shows that the majority of loci genes are fixed for these characteristics.
The leaf number trait is indicative of the genetic material"s environmental and commercial adaption commercialization (Diamante et al. 2013), and improvement of this trait is of paramount importance for lettuce, since its leaves are the commercial end product.There were differences between the progenies; only six of the 11 evaluated progenies were significant underperformers when compared to the other progenies and the cultivars (Table 2).
Among all progenies, 13 were shown to satisfy both the commercial standard and showed significant resistance to M. incognita spp.. Furthermore, the progenies AFX-020C-06-13-02, AFX-020C-06-20-03, AFX-020C-06-20-02, and AFX-020C-06-20-04 originated from families considered segregating for M. javanica (Ferreira et al., 2011).In addition, the same behavior for resistance was found by Gomes et al. (2000) in a mixture of progenies from these families when tested in F 4 and F 5 for resistance to M incognita spp.. Finally, Ferreira et al. (2011) evaluated these progenies for M. javanica resistance and found six resistant progenies, suggesting that maybe the control is found on the same locus.
In this work, 13 progenies were found to be resistant for all the evaluated M. incognita spp.traits.Therefore, these results confirm the hypothesis that the same locus controls resistance to M. javanica and M. incognita spp.However, these progenies need to be evaluated for races 1, 2, 3 and 4 to confirm these results, because the assessments made in this work, and also by Gomes et al. (2000), were done with a mixture of races without knowing for certain what races were involved.
With regard to the visual gall index (Ferreira et al. (2011), this trait is of paramount importance for the genetic improvement program, as it directly affects the lettuce seedling"s health.Resistance for this trait permits a continuation of the program, because it allows a plant to grow and produce seed.In general, for both commercial characteristics and resistance, the progeny AFX-018D-02-23-15-01 stood-out for further cultivar testing.

Table 1 .
Mean square for the average of the lettuce strains in Experiment 1.
Lettuce strains: advanced lettuce cross strains [("Regina 71" × "Grand Rapids") × Elisa]; DP= Plant diameter; HP= Plant height; NL= Leaf number; FML=Fresh mass of leaves; FMP= Fresh mass of plant; LS= Stem length; DS= Stem diameter; BL= Leaf blade; EL= Leaf edge; CL= Leaf color The means followed by different lowercase letters between the genotypes differing by Scott-Knott test (P ≤ 0.05) offspring using the Dunnett test (P < 0.05).The lack of significance between the offspring and the cultivar Grand rapids (resistant) and the significance in relation to Regina-71 (susceptible) for all characteristics, identify the offspring as homozygous resistant.On the other hand, no significant difference between the progeny and the cultivar Regina-71 (susceptible), and the significance in relation to the cultivar Grand Rapids (resistant) for all characteristics, identify those offspring as susceptible.Other observed situations were still considered segregating.For experiments 1 and 2, analyses of variance were carried out and significance after the Ftest means were obtained and grouped by Scoot-Knott test (P < 0.05) and analyzed with the aid of the GENES program

Table 2 .
Mean square for the average of the lettuce strains used for Experiment 2.

Table 3 .
Comparison of mean scores for visual gall index (GI) after inoculation with M. incognita eggs.

Table 4 .
Comparison of mean gall/root system (GL) after inoculation with M. incognita eggs.

Table 5 .
Comparison of the mean eggs/root system inoculated with M. incognita eggs.