suceptibility of Delia platura to seven entomopathogenic nematode isolates from the Central Andes region of Colombia

The seed maggot, Delia platura, is a major pest of spinach crops in the savanna of Bogotá. In Colombia, chemical insecticides are used to manage the pest; however, because its management is not integrated, information about pest management in spinach is still undetermined. Here, we evaluated the susceptibility of D. platura to seven species of entomopathogenic nematodes from the central Andean region of Colombia. Additionally, under laboratory conditions, we produced and evaluated different doses of infective juveniles (IJs) of the most virulent species. In the laboratory, we used yellow potatoes (Solanum phureja) for breeding to obtain third instar larvae; we then exposed them to infective IJs 2500/species. Once we selected the most virulent species, we exposed D. platura to 500, 1000, 2000, 4000 and 8000 IJs/ larvae. We obtained the best results with Steinernema sp.3 with mortality of 75-88% at doses of 4000-8000 IJs, and found that DL50 is 1314 JIs/larvae and DL95 is 15259 JIs/larva. We also evidenced the successful reproduction of Steinernema sp.3 in D. platura, with a mean production of 670±7.67 JIs/larvae for eighteen days. Thus, the seed fly is highly susceptible to Steinernema sp.3 making this species a potential controlling agent for this pest.


introduction
The seed fly Delia platura, Meigen (Diptera: Anthomyiidae), is a cosmopolitan pest that affects more than 40 species of plants such as spinach (Spinacia oleracea L.), artichoke (Cynara scolymus L), beet (Beta vulgaris L) and wild cabbage (Brassica oleracea L.) among others (Darvas andSzappanos 2003, Valenciano et al. 2004).D. platura, is widespread; it is a major pest in Europe, North and South America, and very common in northern Africa, Japan, India, Australia and New Zealand (Valenciano et al. 2004).This dipteran attacks during germination reducing emergence suceptibility of Delia platura to seven entomopathogenic nematode isolates from the Central Andes region of Colombia and causing economic losses affecting up to 30% of production (Valencia et al. 2004).The life cycle of the fly is synchronized with the sowing and harvesting stages.During sowing, the females, attracted to the organic matter, oviposit approximately 100 eggs in the soil near the stems (Capinera 2001, Gouinguene andStadler 2006); then, during germination, the eggs hatch and the first instar larvae feed on seedlings.As the larvae erode the roots and stems, the plants rot, turn yellow and die (See et al. 1975).Larvae feed on plant bulbs and shoots during the harvest, causing great economic losses to producers (Valenciano et al. 2004, Morelock & Correll 2008).
In Colombia, the seed maggot mainly affects spinach crops; it reduces annual production between 5-30%, which represents a loss of between 85.5 and 513 ton/ha (Gil et al. 2007), and is normally controlled using broad-spectrum organophosphate insecticides such as Monitor, methamidophos 600 (not specific to D. platura).However, the inadequate use of pesticides and eventual resistance developed to them by the pest, their high cost (3% of production), and high toxicity and a lack of acquaintance with the natural enemies present in the Cota, Cundinamarca area have led to the exploration of new management plans.Among these plans is biological control using entomopathogenic nematodes given their seeking and specificity capabilities, and their wide host range and reproduction in the field (Valenciano et al. 2004, Gil et al. 2007, Delgado and Saenz 2012).
Nematodes Steinernema spp.Travassos (Rhabditida: Steinernematidae) and Heterorhabditis spp.Poinar (Rhabditida: Heterorhabditidae) are obligate pathogens of insects associated with Enterobacteriaceae bacteria of the genus Xenorhabdus Thomas and Poinar, and Photorhabdus Thomas and Poinar, respectively.Once the nematodes have entered the host's (mouth, anus, spiracles, cuticle), the bacterium spreads producing toxic substances, which cause septicemia and the subsequent death of the insect (Nielsen 2003, Sáenz 2005and Estrada 2008).Nematodes develop within the cadaver feeding on the tissues metabolized by the bacteria.If the infection is successful, the body of the insect can support the propagation of new infective juveniles in amounts proportional to the size of the host (Sáenz 2005, Stuart et al. 2006and Estrada 2008).
Recently, the use of entomopathogenic nematodes (EPNs) has been considered a potential alternative to control Delia species (Chen et al. 2003a, Chen and Moens 2003and Nielsen 2003).Laboratory tests have demonstrated that different doses of Steinernema spp.and Heterorhabditis spp.are efficient between 40-60% in the control of Delia radicum (Linnaeus) (sister species of D. platura).However, the results are inconsistent in terms of the larval stage, nematode species, strain, dose and experimental conditions (Bracken 1990, Royer et al. 1996and Willmott et al. 2002).Both the state of development of the host and the nematode species are factors that significantly influence the mortality of Delia spp.(Willmott et al. 2002, Nielsen 2003).
Research to find new controls in a laboratory setting is a standard procedure, essential to test different nematode species on a given host in order to maximize their impact in the field.In Colombia, no research has been conducted using EPNs to control the seed maggot; the most akin are studies conducted by Gil et al. (2007) to establish the different pests affecting spinach.In consideration of this, this laboratory study evaluates the susceptibility of third instar larvae of D. platura to seven species of nematodes from the Central Andean region of Colombia, six doses of EPNs and the production of infective juveniles of the most virulent type.

Materials and methods
Delia platura Breeding: Delia platura adults were captured in Cota Cundinamarca, at an altitude of 2,547 m, and an average temperature of 13.7°C, in a vegetable producing farm named Alcala.We placed sixteen 1500 ml plastic bottles, on the soil, near the plant for 24 hours, and filled each bottle with 2ml of rice masato (fermented beverage) homogeneously coating the bottle walls (Figure 1a).Once captured, we placed 10 Delia platura adults, and 25g of yellow potato (Solanum phureja) supported on three 1cm x 1cm styrofoam squares and wet sand in sixteen 30x30x30 cm plastic breeding boxes.Additionally, and according to the methodology described by Jimenez et al. ( 2010) we also placed 1 ml of rice masato as a food source (Figure 1b).We stored the 16 breeding boxes for 10 days in the biological control laboratory of the Pontificia Universidad Javeriana under controlled conditions of 70% relative humidity, 12 hours light and 12 hours of darkness and average temperature of 14°C until 7mm (2mg) third instar larvae were obtained.

Obtaining entomopathogenic nematodes:
We used seven isolates of nematodes from the Colombian Andean region (Table 1); six of these were obtained, through Convention No. 182 of   1 were used in the bioassays as well as a control using water.For the experiment we used 120-14 cm 3 plastic cups containing filter paper moistened with 5µl of distilled water, 2500 IJs/larvae, 5g of yellow potato as food and a third instar larvae, in order to simulate field conditions, a total of 15 replicates per treatment, arranged in a completely randomized design.The cups were stored under controlled conditions at a temperature of 20±2°C, and 70% relative humidity.Following the methodology by Kaya andStock (1997), Willmott et al. (2002), and Chen and Moens (2003), we evaluated mortality percent of the larvae in each treatment every 24 hours.The test was repeated three times.

Production of infective juveniles (iJs)
in D. platura larvae: Because its mortality percentages were above 70%, we evaluated the production of IJs for the Steinernema sp.3 isolate.As recommended by Kaya and Stock (1997), 20 D. platura third instar larvae were infected with 2000 JIs/larvae, and incubated at 25°C.Upon completion of the eight-day nematode life cycle, the cadavers were transferred to modified white traps to recover IJs.The cadavers were counted directly using 10 aliquots per sample every 24 hours, until the depletion of the cadavers, following recommendations by Delgado and Sáenz (2012).

Steinernema sp.3 dose:
During the bioassay, to determine the recorded dosis presenting the highest mortality we used Steinernema sp.3 to evaluate the same doses used for D. radicum (Chen et al. 2003), that is, 0, 500, 1000, 2000, 4000, 8000 IJs/larvae.We used 14 cm 3 plastic cups containing filter paper moistened with 5µl of distilled water, 1μl nematode dose, 5g of yellow potato as food and a third instar larvae, for a total of 20 replicates and 5 repetitions per treatment.We stored the experimental units under controlled conditions at a temperature of 20±2°C, and a 70% relative humidity.The larval mortality for each treatment was evaluated every 24.Similary, we evaluated the LD 50 and LD 95 the test was repeated three times.
statistical Analysis: Mortality was converted to a percentage and presented as an average ± the standard error: Unprocessed data met normality (Shapiro-Wilk and Kolmogorov-Smirnov), homogeneity (Levene) and independence (independence test) assumptions, they were therefore analyzed using a one-way ANOVA.To identify which species had the highest mortality and the most effective dose for control of D. platura, we analyzed the tests showing significant differences between treatments retrospectively using a Tukey test (parametric) at a significance level of p ≤0.05, the differences are indicated in the figures with different letters on the bars.We calculated LD 50 -LD 95 values using the Probit analysis.The tests were performed using SPSS 19 software.
Production of iJs in D. platura third instar larvae: IJ production in larvae of D. platura was evaluated using Steinernema sp.3; this isolate presented the highest mortality (>70%).The production of IJs occurred from the third and fourth day, reaching 100% of the production from the ninth to the eleventh day (Figure 3).The average production of IJs of Steinernema sp.3 in D. platura third instar larvae was 670±7.67.
The maximum number of IJs possible in the larvaes was 1.125±10,550 and the minimum was 300±7.67IJs/larvae, and only one generation of EPNs inside them.

Discussion
There have been no previous studies in which these Colombian isolates have been tested on D. platura.We found that the seed fly was susceptible to the Steinernema and the Heterorhabditis isolates; four of the seven (Steinernema sp.1, Steinernema sp.2, Steinernema sp.3, Heterorhabditis bacteriophora) species produced 50-80% larval mortality with respect to the control; Steinernema sp.3 being the most virulent (80%) for

D. platura. Willmott et al. (2002) and Chen & Moes
(2003) evaluated various species of larvae EPNs in D. radicum; their results showed that Steinernema feltiae (Filipjey) is more virulent (45%) than H. bacteriophora Poinar (0-16%), unlike the results from this study in which species of Heterorhabditis spp.only caused 40% mortality.This difference in virulence of EPN species may be explained by pathogenicity and the EPNs' ability to penetrate and avoid or to suppress the host's immune response (Lewis et al. 2006, Li et al. 2007).
The pathogenicity of nematodes is a complex state of attraction, penetration and multiplication (Lewis et al. 1992, Chen andMoes 2003).The IJs migrate to the host responding to stimuli produced by the host, such as CO 2 , temperature, contact, cuticle, feces, these are in turn associated with nematode foraging strategies: ambushing, cruising.(Griffin et al. 2005;Lewis et al. 2006).Ambushers, such as Steinernema sp.3 are able to position themselves vertically erect, allowing them to affix to a substrate using the lowered surface tension (Lewis et al. 2006).In this position, IJs can actively scan the environment for volatile chemical signs such as CO 2 emitted by hosts; this facilitates finding mobile hosts (Chen et al. 2003b).In contrast, cruising foragers like H. bacteriophora and S. colombiense are able to identify signals associated with the cuticle and stool, allowing them to find sedentary (immobile) hosts, which explains their low mortality (≤50%) in D. platura larvae (Lewis et al., 2006, Delgado andSáenz 2012).Like most dipterans, D. platura larvae continuously move within the substrate; therefore, the most effective way to colonize third instar seed fly larvae is using EPNs with an ambusher foraging strategy (Royer et al. 1996).
The number of IJs that can be produced within a host depends on the amount of IJs applied and the size of the host.Nielsen & Holger (2004) estimate the average production of IJs in a 1-2mg larvae in D. radicum is of 518IJs; these values are similar to those obtained in this study, the average production of IJs in D. platura larvae was 679 IJs/larvae for Steinernema sp.3.That is, third instar larvae can sustain the propagation of EPNs because of their large size and length (Nielsen 2003).Moreover, an increased production of IJs in D. platura larvae occurred from day 6 to day 12, otherwise to reports regarding G. mellonella Linnaeus larvae in which most of the production took place during the first three days and gradually decreasing over time, creating one and two short EPN cycles (Realpe et al. 2007).It can be inferred that only one long EPN cycle be can take place within D. platura larvae.
Susceptibility studies in populations of Delia spp, show a positive relationship between dose and larvae mortality, the higher the dose the higher the mortality (Willmott et al. 2002, Chen et al. 2003a, Nielsen 2003).In this study, 4000-8000 IJs/larvae of Steinernema sp.3 produced the highest mortality (≥60%).We found that the medium lethal dose for D. platura larvae was 1,314 IJs/larvae and 1.4414 IJs/larvae was the maximum lethal dose.Chen & Moens (2003) reported the LD 50 for larvae of D. radicum is 258 IJs/larvae and LD 95 for Steinernema arenarium is 15259 IJs/larvae (Aetyukhovsky); this establishes that IJs doses may vary according to the species of EPNs used.The differences in the doses may be explained by the foraging strategy, recognition and penetration ability of the IJs in the host.
EPN species have different recognition capabilities, low or high, which affect their ability to penetrate a host.Chen et al. (2003b) found that Steinernema carpocapsae Weiser has weak recognition capabilities, inconsistent with its ambusher strategy, which prolong its penetration into the host, requiring of more individuals to generate the same mortality.Similarly, common to dipterans, D. platura has small, protected natural openings, which hinder the entry of IJs in larvae, affecting the IJs penetration ability (Capinera 2001).Therefore, the dose required of Steinernema sp.3 is 8000 IJs/larvae to generate 80% D. platura larval mortality.

Conclusion
D. platura larvae showed greater susceptibility to Steinernema sp.3 isolates using a dose of 8x103 IJs/ larvae.These isolates also produced the highest number of IJs in third instar larvae of the seed fly.Accordingly, in the second phase of the study, this dose will be evaluated in the field to determine its performance in the control of D. platura and its possible incorporation into integrated pest management programs in spinach crops in Cota, Cundinamarca.

Fig 3 .
Fig 3. Production time (days) of IJs of Steinernema sp.3 in third instar larvae of Delia platura.The bar indicates the mean with its SE.

Fig 4 .
Fig 4. Percentage of mortality of third instar larvae of D. platura with six doses (IJs/larva) of Steinernema sp.3.The boxes represent the quartiles in which data is located.The horizontal bar indicates data median.The vertical bar SE.The letters on the top of the bars express the differences at p <0.05 (Tukey).

Table 1 .
Colombian soil nematode isolates evaluated on Delia platura.*Speciesisolated at the Pontificia Universidad Javeriana.2009,bythePontificia Universidad Javeriana and Cenicafé (National Coffee Research Center).According toLopez et al. (2007), the morphological characteristics of the strains are related to the species Steinernema sp.1 in the feltiae group, Steinernema sp.2 in the bicornutum group, and Steinernema sp.3 in the carpocapsae group.The Heterorhabditis sp.SL0708 strain was obtained from the Pontificia Universidad Javeriana.All the species were in 1'000.000IJ foams, stored at 5-8°C.We used one day harvested, fresh nematodes for all the tests in this study.