DETERMINING OF ROOT-KNOT NEMATODE ( MELOIDOGYNE JAVANICA ) DAMAGE FUNCTION FOR TOMATO CULTIVARS

Root-knot nematode (Meloidogyne javanica) management should be partly based on the knowledge of the threshold density, and this value is likely to vary depending on the resistance level of the tomato cultivars. The damage functions based on four initial population densities (Pi) (0, 1,000, 3,000 and 5,000 egg kg of soil) of root-knot nematode were determined in four tomato cultivars. The experiment was performed in completely randomized design with four replications. The results showed that yield responses to Pi were fitted properly by a log-logistic function with three parameters. The most susceptible cultivar was ‘Rutgers’ based on EP50 and EP10 (effective population of nematodes, reducing 50% or 10% of maximum yield or shoot fresh weight respectively) and three others were relatively resistant. EP10 is more applicable than EP50 because 50% yield reduction is unacceptable in most situations. EP10 for yield of ‘Rutgers’, ‘Efialto’, ‘Falat 111’, ‘Gina VF’ was 500, 3,021, 2,998, and 3,000 egg kg of soil, respectively. The correlation coefficients among gall index, egg mass and reproductive factors were positively related. Reproduction factor and root gall indices were greater in ‘Rutgers’ than in the other cultivars (P≤0.05). For ‘Gina VF’ as a relatively resistant cultivar it seemed that increasing of Pi up to 5,000 or more egg kg soil might break its resistance.


Introduction
The root-knot nematode (M.javanica) is one of the pests of tomato, but also infests a wide range of other economically important plants (Carter, 1985;Knight et al., 1997).It has a wide distribution particularly in temperate areas of the world (Jepson 1987;Anonymous, 2002).This species causes root galling in susceptible host plants.The infection cycle begins with hatching of the larva, purposed as the Fatemeh Gharabadiyan et al. 148 second-stage juvenile (J2).Infective juveniles enter the root of a susceptible host plant, forming an attachment to feeding site.After that the nematodes become saccate adult females and produce a gelatinous mass of eggs (Eisenback and Triantaphyllou, 1991).Yields of tomato (Lycopersicon esculentum Mill.) are greatly reduced by infestations of M. javanica in Iran (Barooti and Alavi, 1994).The management of root-knot nematodes has traditionally relied on nematicides and other techniques.Two resistant and tolerant cultivars to M. javanica have been reported in Iran (Mortazavi-Bak and Ahmadi, 2004).Khodaei-Arbat (2009) and Moslehi et al. (2010) reported the resistance of four tomato cultivars to M. javanica at an initial population range (0, 1,000, and 2,000 eggs kg -1 of soil).Yield loss and susceptibility of eight tomato cultivars to natural populations of M. incognita Race 1 in Italy and Holland were assessed and two cultivars were considered as resistant (Darban et al., 2003).In the greenhouse trials, the effects of extracts of leaves and roots of Leucaena leucocephala and Gliricidi asepium each at 80,000 mg/kg and 40,000 mg/kg on Meloidogyne incognita on okra were studied (Adekunle and Akinlua, 2007).Resistances of tomato and pepper cultivars to M. incognita were recognized in Florida (Brito et al., 2004).
Resistance to nematodes is usually defined as a plant's ability to inhibit nematode reproduction (Roberts, 2002).The relationship between crop yield response and initial nematode densities is an important property of a crop resistance.The damage threshold is defined as the initial population of nematodes above which yield is suppressed.The most referred function for plant response to nematode initial population is Seinhorst function, but some other functions like linear or sigmoid may be proper.The primary objective of this research was to compare the damage functions for M. javanica on resistant and susceptible tomato cultivars.The effects of resistance on population densities of M. javanica and on root galling of tomato were also studied.

Material and Methods
The experiments were conducted in 2010 in greenhouse conditions.Plants were maintained in a glasshouse adjusted to 25±3°C.Pots (10 cm in diameter × 20 cm in height) were fumigated with 1.3 dichloropropene to eliminate existing nematodes prior to planting and then were filled with a sterile soil composed of sand 89.1%, clay 70%, silt 3.9%, organic matter 2.3% and at pH 7.7.The stock culture was established by placing an egg mass of the nematodes beneath the root system of a cv.'Rutgers' tomato (Lycopersicon esculentum) seedling.The inoculum for experiments was obtained by extracting eggs and second-stage juveniles (J2) from 2-month-old tomato plant cultures using 1% sodium hypochlorite solution (Hussey and Barker, 1973).Nematode inoculum was prepared by combining infected tomato root fragments and infested soil from greenhouse cultures with pasteurized sand.The inoculum concentration was then determined by enumerating the eggs and J2 extracted from the infested soil by elutriation (Byrd et al., 1976) and centrifugation (Jenkins, 1964).Eggs of root-knot nematode were extracted from egg masses adhering to the root fragments recovered during elutriation with NaClO (Hussey and Barker, 1973) and counted.Sufficiently infested soil was incorporated into each pot to achieve the desired Pi to a depth of 5 cm.Treatments were four initial populations (Pi) of M. javanica: 0, 1,000, 3,000, 5,000 eggs and J2 kg -1 soil and four tomato cultivars ('Rutgers', 'Efialto', 'Falat 111', and 'Gina VF') that were arranged in a factorial experiment in a randomized complete design with four replications.Immediately following infestation of the soil, four tomato seeds were planted in each pot in a single row and were thinned to one plant after emergence.Irrigation and pesticide applications were performed.Population densities of M. javanica were estimated at crop maturity (Pf) within 8 weeks after planting.The second-stage juveniles and eggs were extracted from each sample as described above and counted using a stereo microscope.Each pot was hand harvested at crop maturity and yield per pot was determined.Subsequently, root galling for each pot was determined based on a 0 to 5 scale, with 0 representing no galling and 5 representing > 75% of roots galled (Barker et al., 1985).Regression analysis was applied to determine the relationships between Pi and yield for each tomato cultivar.Yield responses to Pi were determined by fitting a log-logistic model (Ritz and Streibig, 2005) with three parameters (Eq. 1) using the computer program R to estimate the effect of host resistance on the damage threshold.

Results and Discussion
The effect of initial nematode population levels on the yield of four tomato cultivars is shown in Figure 1.The log-logistic function with three parameters was adequately able to explain yield response to nematode initial density (Tables 1-2).Effective population of nematodes that reduces 50% or 10% of maximum yield or shoots fresh weight is indicated as EP 50 and EP 10 , respectively.
The most susceptible cultivar was 'Rutgers' based on EP 50 and EP 10 and three others were relatively resistant.EP 10 is a criterion more applicable for plant improvement than EP 50 because 50% yield reduction is unacceptable in most situations.EP 10 for yield of 'Rutgers', 'Efialto', 'Falat 111', 'Gina VF' was 507, 3,062, 2,929, and 3,383 egg kg -1 soil, respectively (Figure 2).The reaction of tomato cultivars to the nematode based on EP 10 and EP 50 parameters is shown in Figure 3. 'Effialto' had maximum response derived from EP 50 as resistant cultivar.Also, the high level of response based on EP 10 existed in 'Falat 111'.The results in Table 3 showed that there are the correlation coefficients between the nematode and tomato traits.The numbers of galls and egg mass were inversely related with shoot fresh weight, root fresh weight and fruit fresh weight.A positive relationship between gall index, egg mass and reproductive factors was observed.Root-shoot ratio (RS) was not correlated with nematode related parameters.The population growing up to 5,000 nematodes per kg of soil seemed to break the resistance of 'Gina VF' cultivar.With population density of 5,000 nematodes the least performance of 'Gina VF' cultivar was 40% while the two cultivars 'Efialto' and 'Falat 111' showed only 20% loss of cortical function.It seems that by increasing nematode populations over 5,000 per 1 kg of soil cultivars lose their resistance.It is suggested that higher populations should be examined in subsequent studies.RF: reproduction factor, SFW: shoot fresh weight, RFW: root fresh weight, FW: fruit weight, FP: fruit production, SL: stem length, SFWP: shoot fresh weight production, R/S: root-shoot ratio.ns , * and ** : non-significant, and significant at the 0.05 and 0.01 levels of probability, respectively.
Among several models in the studies of plant response to the initial population of root-knot or cyst nematode, the one most appropriate that abundantly has been used is Seinhorst model (Duncan and Ferris, 1983;Ploeg and Stapleton, 2001;Castillo et al., 2001).In the Seinhorst's equation, yield in percent from nematode free control and initial nematode population is in logarithmic scale.The model is of the form: y = y m , for x≤ t, ( 1) Where y = crop yield or other plant growth parameter; x = the nematode population density (Pi); t = the nematode population below which yield reduction is not considerable (i.e. the tolerance limit or damage threshold density); y m = mean crop yield where the nematode density is below the tolerance limit (t); m = a constant, usually between zero and one, such that y m × m is the yield at the highest possible nematode density; and z = slope (value between zero and one).Calculation of the Seinhorst equation that best fits the experimental data is rather difficult and time-consuming because the model is segmented (Viaene et al., 1997;Szabo and Somogyi, 2002).
However, other models such as linear forms have also been reported in some studies (Haji-Hassani et al., 2010).Grain yield loss reached a maximum loss of 48% with an initial population density of 20 eggs and J2 g -1 soil reduced in linear trend by cyst nematode (Heterodera filipjevi).Another study is adequately described by linear models, where final population (Pf) increased linearly with Pi (0-3,000 cysts/pot) Heterodera avenae (Al-Hazmi et al., 1999).The selection of a suitable model is dependent on the nature of nematode damage, its initial population, crop tolerance level and agronomic and environmental conditions.Therefore, in a limited range of the initial population, the plant response may be linear, but by increasing the pest density, it might shift to nonlinear trend that means resistance has been broken.In the very high population, the curve slope should approach zero (Schomaker and Been, 1998).
However, in the current study, data did not correspond well to Seinhorst model.Due to cultivar differences in the index of plant growth, the reaction would be very different in variant levels of nematode population density and cannot be measured as a criterion alone (Moore et al., 1988).The results of this study showed that general process could not be considered for shoot fresh weight of each cultivar.These results have compatibility with studies (Moore et al., 1988) to evaluate resistant varieties of beans to nematode that also emphasized this fact as well.However, the response of each cultivar at each level of nematode population compared with the control treatment is considered to be separate.In this regard, in the cultivar 'Falat 111' there was no significant difference between population levels (0, 1, 3, 5 egg and J2 per g of soil) relative to control treatment.The shoot fresh weight of the 'Efialto' cultivar decreases with the nematode population increase.It seems that low population causes an increase and high population causes a decrease in the physiological activity of the plant (Bird, 2004).Comparing the number of galls and nematode population levels for each cultivar the results show that 'Rutgers' at all levels has shown the most significant difference in comparison to other cultivars.Gall index is one of the important factors for evaluating the resistance.'Rutgers', as the most sensitive cultivar produces a lower yield and has weaker growth parameters than other cultivars even in root-knot nematode free plots.This may mean that relatively resistant cultivars have a good performance in both controls and infected trials.
The results of this study show compatibility with previous experimental results (Tood et al., 1991) to evaluate the resistance of cucumber.It can be said that cultivars with increased competition between larvae to penetrate the root and form the feeding site lead to reduction of positive introductions in root and subsequent decrease of the number of galls (Divito and Greco, 1998).Hypertrophy and hyperplasia can also occur together as plant response to nematode attack (Endo, 2003).Inoculum level produced quantitative differences in resistance for two species of Meloidogyne (Araujo et al., 1982).Host parasite relationships and pathogenicity of Meloidogyne javanica on potatoes were studied under glasshouse and natural conditions and showed the same results (Vovlas et al., 2005).
High initial population leads to the increase in the number of egg mass.Singh and Khurma (2007) also showed that increasing the inoculum concentration enlarges the average number of egg mass in the roots of infected plants.According to the results there is not a necessary relationship between galls and egg masses.This finding is consistent with previous results (Olowe, 2007;Udo et al., 2008) in the evaluation of resistance and sensitivity in cucumber cultivars to root-knot nematode.Stimulation of primary root gall may only come into existence by nematodes, but they are not able to complete their life cycle on the resistant plant (Williamson and Hussey, 1996).The high reproduction rate was observed in 'Rutgers' as a sensitive cultivar.Final population of root-knot nematode in the soil increased in susceptible cultivars (Khan et al., 2000).There is no necessary relationship between gall number and final population of nematodes (Trudgill and Blok, 2001).All in all, the results of these experiments showed that the logarithmic equation-logistics in resistant and susceptible cultivars could offer useful information on the processes of change of the the plant growth characteristics with different Meloidogyne javanica nematode populations measured.

Conclusion
The purpose of the current study was to determine damage function for some resistant and susceptible tomato cultivars.This project was undertaken to design by a log-logistic function with three parameters and evaluate the correlation coefficients among gall index, egg mass and reproductive factors that were positively related.Reproduction factor and root gall indices were greater in 'Rutgers' than in the other cultivars (P ≤ 0.05).For 'Gina VF' as a relatively resistant cultivar it seemed that increasing of Pi up to 5,000 or more egg kg -1 soil might break its resistance.It was also shown that the most susceptible cultivar was 'Rutgers' based on EP 50 and EP 10 and that three others were relatively resistant.

Figure 1 .
Figure 1.Fruit fresh weight response (percentage of root-knot free control) to initial population of M. javanica.

Figure 2 .
Figure 2. Shoot fresh weight response (percentage of root-knot free control) to initial population of M. javanica.

Figure 3 .
Figure 3. EP 10 and EP 50 parameters of different tomato cultivar response to M. javanica.

Table 1 .
Parameters and standard errors of the fitted equations of the nematode population affected yield of tomato cultivars.and ** : significant at the level of probability 0.05 and 0.01 respectively. *

Table 2 .
Parameters and standard errors of the fitted equations of the nematode population affected shoot fresh weight of tomato cultivars.

Table 3 .
Correlation coefficients between different traits in tomato cultivars.