Advances in Applied Agricultural Sciences 2 (2014); 10: 09-21
Can Trichogramma and entomopathogenic nematodes serve as effective biocontrol options for Helicoverpazea in tomato?
Atwa A. Atwa
Plant Protection Research Institute (PPRI), Agriculture Research Center (ARC), Dokki 12618, Giza, Egypt.
Biocontrol for insect pest management (BIPM) was the maiden like method of pest control pre-dates the modern pesticide era. Controlled exotic pests by natural enemy species collected from the country or area of origin of the pest was one of the successful method in for BIPM. The potential of entomopathogenic nematodes (EPNs) and an egg parasitoid for biological control of eggs of tomato fruit worm Helicoverpa (= Heliothis) zea (Boddie), and the effect on fruit production after two years of field evaluation (2011-2012) revealed encouraging results. Trichogramma evanescens (Westwood), Steinernema carpocapsae (all strain) (Weiser) and Heterorhabiditis bacteriophora (HP88) (Poinar) were applied to control H. zea on tomato field. Two augmentation releases of each biological control agent at 15-day intervals, 15 days after tomato plants reach 30-50% effloresce were found effective. The percentage of fruits damaged by H. zea was significantly reduced in plots treated by egg wasp T. evanescens than either of S. carpocapsae or H. bacteriophora nematodes and control treatments. The efficacy of T. evanescens, S. carpocapsae, and H. bacteriophora differed significantly and S. carpocapsae nematode was found superior to H. bacteriophora. The healthy fruit yield in plots treated by H. bacteriophora was better than the fruit yield in plots treated by T. evanescens and S. carpocapsae. While as the average yield from healthy fruits/plants in plots treated by H. bacteriophora, T. evanescens and S. carpocapsae was better than the numbers in control plots. It was conducted that biological control in tomato for controlling H. zea using Trichograma and EPNs as one of the most promising alternatives to the present chemical based pest management. These biocontrol agents (H. bacteriophora, S. carpocapsae & T. evanescens) are easy to apply. Coast effective compared chemical pesticides, and turned out to be safe with high quality fruits.
Lepidopteran insect pests are one of the important constraints to tomato production in Egypt, in particularly the tomato fruitworm, Helicoverpa (=Heliothis) zea (Boddie), which cause direct and indirect yield loss as aesthetic damage (Atwa, 2009). Tomato fruit worm, H. zea is one of the most devastating insect pests of agriculture in Egypt, attacking wide range of cash and subsistence crops. It is a serious insect pest of cotton, corn and tomatoes (Luttrel, 1994) as its several common names indicate (e.g., bollworm, corn earworm, tomato fruit worm). It is also injurious to beans, cabbage, lettuce, pepper, alfalfa, clover, vetch, tobacco and other cultivated crops. The moths emerge during the spring and early summer and, after mating, deposit their eggs singly at dusk on the plants on which the larvae are to feed. Each moth lays from 500 to 3000 eggs, averaging about 1000, which hatch in 2 to 11 days.
During fruiting phase, the tomato fruitworm, H. zea will complete its larval stage inside the tomato fruit. Early stage larvae enter fruit at the stem end when it is between 2-5 cm in diameter. The larvae feed for 14 to 28 days after which they burrow into the soil and pupate (Metcalf et al., 1962). H. zea larvae may feed on foliage and burrow in the stem of tomato, but most feeding occurs on fruits. Larvae commonly begin to burrow into fruits. Tomato is more susceptible to injury, before corn is silking. In the presence of corn, moths will preferentially oviposit on fresh corn silk. During larval development, caterpillars may move from one fruit and enter another. Their feeding results in a messy, watery, internal cavity filled with cast skins and feces. Damaged fruit will ripen prematurely. Late in the season, small larvae will also enter ripe fruit. Small larvae are difficult to detect and, thus, may be a problem in processing tomatoes for the canner. Management of the tomato fruit worm is usually achieved by application of insecticides. In general, the use of insecticides and other chemical treatments implies the risk of adverse ecological, toxicological and economic effects. Alternative techniques – mainly biological control include the use of entomopathogenic nematodes and insect egg parasitoids (Atwa, 2009) are one of the most promising alternatives to chemical pesticides in pest management. Although traditionally it has been focused on introduction and permanent establishment of natural enemies, greater effort is now being directed toward augmentative, particularly toward inundative, biological control (Mills, 1990; Parrella et al., 1992).
Trichogramma species are among the most commonly used groups of natural enemies because they are relatively easy to mass rear and, being egg parasitoids, which kill the host before crop damage occurs. However, the effectiveness of Trichogramma inundation has been variable (Li, 1994). One reason may be the parasitoid release without proper knowledge on the biology and the population dynamics of the selected species. The use of polyphagous egg parasitoids of the genus Trichogramma for the control of eggs of various moth species of orchard and field crops has received much attention (Parker & Pinnell, 1972; Ridgway & Vinson, 1977). Very large numbers of Trichogramma adults are required for inundative releases to suppress established populations of moths in field crops of orchards. The egg parasitoid, Trichogramma evanescens is extensively used in inundative releases against a number of lepidopterous pests in Europe (El-wakeil & Hussein, 2009; Tran & Hassan, 1986).
EPNs are used to control a variety of economically important insect pests such as some lepidopterous insects (Atwa, 1999). The genera of Steinernema and Heterorhabditis are obligate parasites of insects (Poinar, 1990; Grewal et al., 2005). These EPNs have a mutualistic symbiosis with a bacterium (Xenorhabdus spp. and Photorhabdus spp. for steinernematids and heterorhabditids, respectively) (Poinar, 1990). Infective juveniles (IJs), the only free-living stage, enter hosts through natural openings (mouth, anus and spiracles), or in some cases, through the cuticle. After entering the host’s hemocoel, EPNs release their bacterial symbionts, which are primarily responsible for killing the host within 24-48 h, defending on secondary invaders and providing the EPNs with nutrition (Dowds & Peters, 2002). The EPNs complete up to three generations within the host after which IJs exit the cadaver to search for new hosts (Kaya & Gaugler, 1993).
The use of commercial strain of T. evanescens, EPNs (S. carpocapsae “all strain” and H. bacteriophora “HP88” strains) as biological control agents for suppression of the tomato fruit worm, H. zea are being evaluated in Egypt before by Atwa 2009. In view of the above facts the present study was conducted to assess the efficacy of used different types of biocontrol agents Trichogramma and EPNs under field conditions for the management of fruit borer in Tomato and changes in quality and quantity fruits production.
Materials and Methods
Field experiment design:Four plots of tomato field plantation (three for treatments and one for control), each 400 m2 of ca. 900 plants/plot were selected at Meet-Kados region, Giza Governorate for each treatment. The experimental plots were grown on 25 December with tomato cultivar; Solanumlycopersicum(=Lycopersicum esculentum) (L.) “GS variety”. Three treatments (T. evanescens, H. bacteriophora & S. carpocapsae) and control experiment were used in this study. Plots were separated from each other by 5 rows of untreated tomato plants. A randomized complete block design incorporated 3 plots for each treatment was established (Atwa, 2009). Tomato plants reach 30-50% effloresce by mid-March and treatments by EPNs and T. evanescens were performed twice for field released after 15 days and 30 days of 30-50% tomato effloresce (Atwa, 2009). The experiment was conducted during two consecutive growing seasons (2011-2012). Local and commercial practices were followed to grow tomato plants, at the same time no insecticides used during the whole period of the present study (Atwa, 2009, Hegazi et al., 2012).
Entomopathogenic nematodes used in experiment: Heterorhabditis bacteriophora (HP88 strain) and Steinernema carpocapsae (all strain) used in this study were cultured on the last instar larvae of Galleria mellonella (L.) according to the method of Dutky et al. (1964). Entomopathogenic nematodes (EPNs) infective juveniles (IJs) belong to H. bacteriophora and S. carpocapsae were harvested from nematode traps as described by White (1927) at 25+2oC. A stock suspension of the IJs was stored at 15oC in sterilized distilled water until used in field experiments. All nematodes were used within 2 weeks of harvest and a new infection cycle and a stock of IJs was prepared every 2 weeks.
Preparing Trichogramma for field release: Commercially available species of T. evanescens was used for field releases. The wasps were reared at the Department of Entomology, Faculty of Agriculture, Alexandria University. The original isolate of T. evanescens was isolated from eggs of Chilo agamemnon Bles on sugar cane (Ahmed & Kira, 1960; Abbas, 1990). T. evanescens is arrhenotokous species with a female-biased sex ratio of 60-70% females (Pintureau et al., 1999). A small cardboard cards (1.5×3.5 cm) were prepared by gluing 3000-4000 (depending on expected sex ratio) parasitized eggs of Sitotroga cerealella using for field releasing. Eggs contained parasitoids of different developmental stages to ensure a staggered emergence for a continuous presence in the field (Atwa, 2009).
Table 1. Tomato fruit infestation (mean percentage) due to H. zea infection in different treatments during 2011 and 2012seasons
H. zea infection 2011
H. zea infection 2012
Table 2. ANOVA table for comparative analysis of total effect EPNs and Trichogramma on average of infected fruits
Table 3. Variation in numbers of tomato fruits production in different treatments of bio-agents during 2011 and 2012
Numbers of tomato fruits production 2011
Numbers of tomato fruits production 2012
Table 4. ANOVA table for comparative analysis of total effect of EPNs and Trichogramma on average of healthy fruits production
Fig. 1. Percentage (Mean ± SD) of infected tomato fruits by H. zea larvae during various inspection dates (A: 2011 & B: 2012) of fruit maturation in treated plots by Trichogramma evanescens.
Mass releasing:The release of both EPNs and Trichogramma were repeated only twice in this experiment. On the EPNs release plots, both EPN species (H. bacteriophora & S. carpocapsae) were applied with concentration of 15000 infective juveniles/plant or 30000 IJs/m2 (about 13.5×106/plot). The application was done before sunset using 10 liters portable spraying. 50 ml of super film was added to the nematodes suspension (Atwa, 2009). While as T. evanescens release plots, an application rate of approximately 12000 female wasps per each plot was applied (about 3000 female wasps/card, 6cards/ plot) for each release for the Trichogramma. This application was repeated for twice as mentioned before.
Data collection: Percentage of damaged tomato fruits by H. zea larvae and healthy produced fruits were recorded during the harvesting period from April 15 to May 15 of both growing season (the harvesting was done every 5 days). At each period of harvesting, 20 plants were selected randomly from each plot (i.e., experimental square), 4 from each corner and 4 from the middle, to count the number of healthy and damaged tomato fruits (Atwa, 2009). Then, the plants were marked to ignore them during the next inspection.
Statistical analysis:The data percentage values in this study were normalized using arcsine transformation. The significance of the mean effects was determined by analysis of variation (ANOVA). The significance of various treatments was evaluated by Duncan’s multiple range test (P<0.05) (SAS Institute, 1988). Also all data processing was performed off-line using a software package (MATLAB 8.0) to displays an interactive graph of the estimates with comparison intervals around them.
Evaluation efficacy of Trichogramma and EPNs against tomato fruit worms
The current study was focused on the damage of tomato fruits and total production decrease caused by H. zea infestation. Evaluation of control efficacy using Trichogramma and EPNs against H. zea was demonstrated by evaluation the mean (± SD) of infected tomato fruits (damage fruits) during 2011 and 2012 in Figures (1, 2 & 3). Data in Figures (1.A) illustrated that there is a difference between the control group and T. evanescens treatment in year 2011 due to relative infection. At the same time there is a difference between control group and T. evanescens treatment in year 2012 due to infection (Figures 1, B). Efficacy of S. carpocapsae was more efficacies than control as illustrated in Figures (2). Data in Figures (2.A) showed the difference between the control group and S. carpocapsae treatment in year 2011 due to infestation. At the same parallel there is a difference between the control group and S. carpocapsae treatment in year 2012 due to infection comparative (Figures 2.B). Otherwise data in Figures (3.A) was demonstrated that, there is a difference between the control group and H. bacteriophora treatment in year 2011 due to infection comparative. At the same parallel there is a difference between the control group and H. bacteriophora treatment in year 2012 due to infection comparative (Figures 3.B).
The variation of control efficacy between the two years of experiment and the different treatment was discussed using one way ANOVA analysis in table (1). There was highly significant between the treatments and control treatment during the year 2011 (F=319.484, df=139,556). Using T. evanescens (Mean ± SD = 2.88±2.19) for control H. zea was highly effected than S. carpocapsae (Mean ± SD = 6.1±2.15) and H. bacteriophora (Mean ± SD = 6.23±2.35), comparing with control treatment (Mean ± SD = 9.51±2.39) as mentioned in table (1). As the same parallel the data in Figures (4) was cited the highly significant (F=974.66, df=139,556) between T. evanescens, S. carpocapsae, H. bacteriophora and control treatment due to differences between Mean (± SD) which was (0.38±0.10, 6.13±2.24, 6.58±1.97 and 9.73±2.26) respectively.
Fig. 2. Percentage (Mean ± SD) of infected tomato fruits by H. zea larvae during various inspection dates (A: 2011 & B: 2012) of fruit maturation in treated plots by Steinernema carpocapsae.
Fig. 3. Percentage (Mean SD) of infected tomato fruits by H. zea larvae during various inspection dates (A: 2011 & B: 2012) of fruit maturation in treated plots by Heterorhabiditis bacteriophora.
The all mean of control efficacy for experiment in two years of study was mentioned in Figures (4) and ANOVA table analysis in table (2) (F=444.4, df=3, 556, Prob>F=0, P<0.05). The percentage of damaged fruits by H. zea was reduced in plots of the egg wasp T. evanescens release more than the two EPNs species (S. carpocapsae, & H. bacteriophora) and control treatments. These results in Figures (4) further indicate that the efficacy of T. evanescens, S. carpocapsae, and H. bacteriophora differed significantly. At the same time S. carpocapsae nematode was more potent in decreasing the fruit damage when compared with H. bacteriophora. While as in all trials, application of T. evanescens wasps proved to be more effective than either of S. carpocapsae or H. bacteriophora nematodes (Figures 4).
Fig. 4. Percentage (Mean ± SD) of total infected tomato fruits by H. zea larvae during various inspection in two years of fruit maturation in treated plots by EPNs and Trichogramma compared with control treatment.
Fig. 5. Average numbers (Mean ± SD) of produced tomato healthy fruits due to field application of Trichogramma evanescens for controlling H. zea during various inspection dates (A: 2011 & B: 2012).
Effect of management strategies on tomato fruits production
The indirect effect of population reduction of H. zea in tomato treated plots by EPNs and Trichogramma was measuring the difference of fruits production in treated plots. Data in Figures (5.A) shows that there is a difference between the control group and T. evanescens treatment in year 2011 in number of produced tomato healthy fruits due to treatment of biocontrol agent. At the same parallel, there is a difference between the control group and T. evanescens treatment in year 2012 in number of produced tomato healthy fruits (Figures 5.B). As the same parallel, there is a difference between the control group and S. carpocapsae treatment in year 2011 in number of produced tomato healthy fruits due to treatment of EPN species (Figures 6.A). Otherwise, there is no difference between the control group and S. carpocapsae treatment in year 2012 in number of produced tomato healthy fruits (figure 6.B). Whereas, data in Figures (7.A) illustrated that, there is no difference between the control group and H. bacteriophora treatment in year 2011 in number of produced tomato healthy fruits due to treatment of EPN species. On the otherwise, there is a difference between the control group and H. bacteriophora treatment in year 2012 in number of produced tomato healthy fruits (Figures 7.B).
Difference of tomato fruit production through the two years of experiment and the different treatment was discussed using one way ANOVA analysis in table (3). Highly significant between the treatments and control treatment during the year 2011 (F=3.845, df=139,556). Significant differences was shown in table (3) while the numbers of healthy fruits in plot treated with H. bacteriophora (Mean ± SD = 32.0089±4.4771) was more than the numbers of healthy fruits in plots treated by T. evanescens (Mean ± SD = 31.0933±4.9430) and S. carpocapsae (Mean ± SD = 28.3422±4.1118) comparing with control treatment (Mean ± SD = 26.6178±4.1526). ANOVA analysis variation indicates highly significant variation in treated plots by EPNs and Trichogramma (table 3) during 2012 (F=7.933, df=139,556). The Mean ± SD for healthy fruit numbers in plots treated by T. evanescens, S. carpocapsae, H. bacteriophora and control treatment was 29.8800±3.8042, 27.3733±3.6685, 31.1244±4.2186 and 27.3822±4.1074respectively.
Total mean numbers of tomato healthy fruits during two years of study in all plots of experiments shows significant differences (F=21.28, df=3, 556, Prob>F=1.51212e-013, P<0.05) as illustrated in table (4). Data in Figures (8) shows the differences in average numbers of produced healthy fruits/plant. Data in Figures (8), shows that average numbers of produced healthy fruits in plots treated by H. bacteriophora was better than the numbers in plots treated by T. evanescens and S. carpocapsae. While as the average numbers of produced healthy fruits/plants in plots treated by H. bacteriophora, T. evanescens and S. carpocapsae was better than the numbers in control plots (Figures 8).
Fig. 6. Average numbers (Mean ± SD) of produced tomato healthy fruits due to field application of Steinernema carpocapsae for controlling H. zea during various inspection dates (A: 2011 & B: 2012).
Fig. 7. Average numbers (Mean ± SD) of produced tomato healthy fruits due to field application of Heterorhabiditis bacteriophora for controlling H. zea during various inspection dates (A: 2011 & B: 2012).
Fig. 8. Average numbers (Mean ± SD) of total produced tomato healthy fruits due to field treatment by EPNs and Trichogramma compared with control treatment for controlling H. zea during various inspection in two years of fruit maturation.
Using EPNs (H. bacteriophora & S. carpocapsae) and T. evanescens wasps has proven successful as safety environmental bio-agents than conventional insecticides for controlling the tomato fruit worm (Atwa, 2009). Mean percentages of infested fruits were significantly less in all plots after applying the used biocontrol agents (H. bacteriophora, S. carpocapsae & T. evanescens) in the two successive study years than those from the control group. Applications of T. evanescens were most effective agent and reduced fruit damage to 2.88 and 0.38 during 2011 and 2012 respectively. The outcome of T. evanescens field experiments was encouraging with an efficient use of Trichogramma spp. in controlling the H. zea. These observations were in agreement with Oatman & Platner (1971) who had reported that, the levels of parasitism averaging 40 to 80% have been attained by such releases in California and Florida, resulting in fruit damage levels of about 3% (Atwa, 2009). The host crop seems to affect parasitism rates, with tomato being an especially suitable crop for parasitoid releases (Martin et al., 1976). In fact, the field release of mass-reared egg parasitoids of the genus Trichogramma could be an option (Li, 1994), but has never been properly explored.
Regarding the EPNs (H. bacteriophora & S. carpocapsae) the obtained results revealed appreciable field efficacy of S. carpocapsae against H. zea, resulted in more protection of tomato fruits than those from H. bacteriophora group. The H. bacteriophora was the least effective test agent and was in agreement with (Atwa, 2009). According to Choo et al. (1989) and Alatorre-Rosas & Kaya (1990), H. bacteriophora searches for hosts and generally infects deeper in the soil profile, whereas S. carpocapsae waits and infects hosts near the soil surface. High efficiency of heterorhabditid nematodes was reported against the Japanese beetle, Popillia japonica Newman (Georgis & Gaugler, 1991). In Texas, Steinernema riobravis has been found to be an important mortality factor of prepupae and pupae of corn earworm, but this parasitoid is not yet generally distributed (Alatorre-Rosas & Kaya, 1990). Similarly, Heterorhabditis heliothidis (=bacteriophora) has been found parasitizing corn earworm in North Carolina, but it has not been found widely (Alatorre-Rosas & Kaya, 1990). Both of the latter species are being redistributed, and can be produced commercially, so in the future they may assume greater importance in natural regulation of earworm populations. In the present investigation, the steinernematid nematode was found to be the most promising nematode agent for further studies of controlling the underground stages of H. zea that pupate near the soil surface. S. carpocapsae seems to be a potential bio-control nematodes agent for H. zea. That is agreed with Purcell et al. (1992) whom reported that EPNs provide good suppression of developing larvae if they are applied to corn silk; this has application for home garden production of corn if not commercial production. Finally biological control in tomato for controlling H. zea using Trichograma and EPNs was found to be one of the most promising alternatives to pesticides in pest management.
The plots treated with biocontrol agents for controlling H. zea had significantly high in fruit production than the control treatment. As suggested by van Lenteren (1997) reported that biological pest control was well established in European greenhouse vegetable production (tomato and cucumber), and has been the cornerstone of IPM for the last 30 years, (and) that as well as its environmental benefits, IPM adoption has given European growers significant savings in labour and costs. While as the pesticides are widely used by tomato growers. More sustainable production methods are readily available but, unfortunately, not widely disseminated. We strongly believe that biological control methods and improved cultural practices can reduce pesticide use dramatically. The favorable economics of used biocontrol agents (H. bacteriophora, S. carpocapsae & T. evanescens) relative to the use of pesticides also applies to the commercial application of the technique. Thus the use the use of these biocontrol agents (H. bacteriophora, S. carpocapsae & T. evanescens) were found easy to apply and less in cost comparing to pesticides, and produced high quality safe production of tomato fruits.
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