Identification of suitable Trichogramma sp. and working-out dosages for management of Brinjal Shoot and Fruit Borer under laboratory condition

Brinjal is grown throughout the country, all round the year (Choudhary, 1970) and is believed to be cultivated in India for the last 4,000 years. South East Asian countries account for almost 50 per cent of world’s area under brinjal cultivation. Brinjal assumes special significance among vegetables in the hot wet season when other vegetables are in short supply and it is the only vegetable that is available at an affordable price for rural and urban poor. So, it is known as a poor man’s crop.


Introduction
Brinjal is grown throughout the country, all round the year (Choudhary, 1970) and is believed to be cultivated in India for the last 4,000 years. South East Asian countries account for almost 50 per cent of world's area under brinjal cultivation. Brinjal assumes special significance among vegetables in the hot wet season when other vegetables are in short supply and it is the only vegetable that is available at an affordable price for rural and urban poor. So, it is known as a poor man's crop.
Brinjal is attacked by a plethora of insect and mite pests, starting from seedling stage to senescence. A survey carried out by the Asian Vegetable Research and Development Centre (Anon., 1995) indicated that the brinjal shoot and fruit borer (BSFB), Leucinodes orbonalis Guenée, leaf hopper, Amrasca biguttula biguttula Ishida and epilachna beetle, Henosepilachna (Epilachna) vigintioctopunctata Fabricius are the most destructive pests on brinjal in Asia. Independently, in the entire South Asian region, the shoot and fruit borer has been identified as the primary limiting factor in brinjal production. Occasionally, brinjal is severely infested by mites, Tetranychus sp., aphids, Aphis gossypii Glover and whiteflies The shoot and fruit borer, Leucinodes orbonalis has a countrywide distribution and has been categorized as the most destructive and most serious pest causing huge losses in brinjal. In the present investigation, six species of Trichogramma were used for selection of most suitable species against shoot and fruit borer, L. orbonalis, the per cent parasitism ranged between 3.60 to 93.20 per cent and the highest per cent parasitism was observed in T. evanescens (93.20 %), which was on par with T. chilonis 92.00 per cent. In a dosages experiment, the highest per cent parasitism of 74.1 per cent was recorded in the dosage having 15 adults/sq. m (150000 adults/ha) and lowest parasitism of 44.2 per cent was recorded in dosages of 5 adults/sq. m (50000 adults/ha). Among the two species highest parasitism was recorded in the T. chilonis (68.0 %) compared to T. evanescens (55.9 %) and 100,000 was found to be optimum dosages.
The shoot and fruit borer is reported to be parasitized by number of parasitoids (Krishnamoorthy and Mani, 1998), among them, Trathala flavor-orbitalis was recorded as one of the important parasitoids causing considerable larval parasitization.
The information on seasonality of natural enemies is scanty, particularly their availability in sprayed and unsprayed conditions. However, no attempts have ever been made to explore, mass multiply and utilize parasitioids under field conditions for extensive control of BSFB in India. No attempt has been made to determine molecular genetic diversity among different populations. Such information based on existence of haplotypes could pinpoint the availability or effectiveness of natural enemies and devise control programmes.The number of polymorphic markers was higher indicating the diversity among the populations (Lynch and Milligan, 1994;Williams et al., 1990).

Identification of suitable species of Trichogramma spp.
In this experiment, the glass tube having size 70 sq. cm (20 × 3.5 cm) was used for screening of six different species of trichogrammattids against the eggs of BSFB (Table 1). The species selected were based on their record of parasitizing BSFB eggs under the field conditions in different regions or world used for field evaluation.
Fifty Leucinodes eggs laid on black cloth were pasted on 6 × 2 cm paper card with fine layer gum and was placed in the glass tube separately for screening of six different species of trichogrammattids and fine streak of 50 per cent honey was provided as adult food. Parasitoids (@ 1 females/50 eggs of Leucinodes orbonalis) were released in each glass tube 70 sq. cm (20 × 3.5 cm) for parasitism by covering the mouth of the tube by double layered black cloth. The parasitized cards were collected and placed in another glass tube after 24 h of parasitism. After 4 or 5 days, the parasitized eggs turned black and black eggs were counted and recorded per cent parasitism using the formula and sexratio was recorded separately for all six different species of trichogrammattids. The per cent parasitism was calculated by using the formula: Number of black eggs with emergence hole % Parasitism = -----------------------------× 100 Number of host eggs provided for parasitism From the above experiment, the species which gave the highest per cent parasitism was selected for cage study to workout dosage and finally select most efficient species for field evaluation. The study was conducted in insect rearing cage (1 sq. m) made up nylon mesh with front opening zip. The two selected Trichogramma species, T. chilonis and T. evanescens were tested at three different dosages, viz., @ 50,000, 100,000 and 150,000/ha (Table 3). The eggs of L. orbonalis were used and parasitoids in the three dosages were released at the rate 5, 10 and 15 adults for cage of 1 sq. m size. The eggs offered for parasitism was at the rate of 250 eggs/treatment and the experiment was replicated 10 times.
To check the parasitism at different treatment dosages, the Leucinodes eggs were counted and pasted on the 6 × 2 cm paper card with fine layer gum and was placed inside in the top of the cage and fine streak of 10 per cent honey was provided as adult food. Parasitoids (@ 5, 10 and 15 adults/250 eggs of Leucinodes) were released in cage having size of 1 sq. m. The parasitized cards were collected and placed in glass tube after 24 h of exposure in the cage.
After 4 or 5 days the parasitized eggs that turned black were counted and recorded for per cent parasitism was recorded using the formula as described above and sex-ratio was recorded separately for two different species of Trichogramma. From this experiment, most promising species was selected that gave highest parasitism and dosages was also decided. The selected species was used for field trial at most effective dosage. The data is analyzed by using the two-way ANOVA for interpretation of final selection of species and dosage based on CD values.

Screening of various trichogrammatids against brinjal shoot and fruit borer, L. orbonalis in test tube
The six species of trichogrammatids like Tr. bactrae, Tr. armigera, T. achaeae, T. japonicum, T. chilonis and T. evanescens were used for screening of Leucinodes eggs for selecting the most suitable species for further experiments (Table 1).

Per cent parasitisation
The screening of different trichogrammatid species revealed that the per cent parasitisation ranged between 3.60 to 93.20 per cent and the highest was observed in T. evanescens i.e., 93.20 per cent, which was on par with T. chilonis i.e., 92.00 per cent, followed by T. achaeae, Tr. armigera, Tr. bactrae, whereas T. japonicum gave lowest per cent of parasitisation of 3.60 per cent (CD = 3.19, P = 0.05) ( Fig. 1; Table 2).

Per cent adult emergence
The exposure of L. orbonalis eggs for trichogrammatid species the adult emergence ranged from 7.60 to 95.60 per cent and the highest per cent adult emergence was observed in T. chilonis i.e., 95.60per cent, which was on par with T. evanescens i.e., 94.40 per cent, followed by T. achaeae, Tr. armigera, Tr. bactrae and least in case of T. japonicum i.e., 7.60 per cent (CD = 4.63; P = 0.05) ( Fig. 1; Table 2).

Per cent female emergence
The per cent female emergence ranged from 2.80 to 65.20 per cent, the highest emergence was recorded for T. chilonis i.e., 65.2 per cent, which was on par with T. evanescens i.e., 62.4 per cent, followed by T. achaeae, Tr. armigera, Tr. bactrae and the least was by T. japonicum i.e., 2.80 per cent (CD = 0.3.53; P = 0.05) ( Fig. 1; Table 2).

Screening of trichogrammatids at different dosages against brinjal shoot and fruit borer, L. orbonalis eggs under caged condition
From the above screening experiments, two species of trichogrammatids, viz., T. chilonis and T. evanescens were selected for determination of their optimum dosages for field evaluation.

Per cent parasitisation
The two factor ANOVA analyses revealed that there is a significant difference between the dosages used and the species selected with respect to per cent parasitisation. The highest per cent parasitism of 74.1 per cent was recorded in the dosage having 15 adults/sq. m (150000 adults/ha) and lowest was recorded in dosage having 5 adults/sq. m (50000 adults/ha) with parasitisation being 44.2 per cent. But the per cent mean parasitisation among the two species was highest in T. chilonis (62.50 %) as compared to T. evanescens (57.70 %) and it differed significantly (CD = 4.81; P = 0.01) ( Table 3).

Per cent adult emergence
The results revealed that there was a significant difference between the dosages used and the species selected with respect to per cent adult emergence.
The highest per cent adult emergence of 72.85 per cent was recorded in the dosage having 15 adults/sq.m (150000 adults/ha) and lowest was observed, in case of dosage having 5 adults/sq.m i.e., 43.10 per cent (50000 adults/ha).
The per cent adult emergence among the two species was highest in T. chilonis (61.4 %) as compared to T. evanescens which showed 56.6 per cent (CD = 4.81; P = 0.01) ( Table 3). In general highest emergence was recorded at the highest dosage.

Per cent desiccation
The results revealed that there were nonsignificant differences between the dosages evaluated and the species selected with respect to per cent desiccation, by two factor ANOVA analyses (Table 3).

Per cent female emergence
The results revealed that there was a significant difference between the dosages used and two species with respect to per cent female emergence by two factor ANOVA analysis. The highest per cent female emergence recorded was 36.75 per cent in the dosage having 15 adults/sq.m (150000 adults/ha) and lowest per cent female emergence of 22.10 per cent was recorded when dosage was 5 adults/sq.m (50000 adults/ha) (Table 3). Nevertheless, the per cent female emergence was highest in case of T. chilonis as compared to T. evanescens.

Screening of various trichogrammatids against brinjal shoot and fruit borer, L. orbonalis in test tube condition
The selection of most promising species or strains of Trichogramma has been advocated as first step in achieving successful control program (Hassan, 1989). In order to select most promising species for evaluation against brinjal shoot and fruit borer, L. orbonalis, six species of trichogrammatids like Trichogrammatoidea bactrae, Tr. armigera, Trichogramma achaeae, T. japonicum, T. chilonis and T. evanescens were used in the present study.
In the present study, the per cent parasitism was ranged between 3.60 to 93.20 per cent, at least two species, T. evanescens and T. chilonis parasitized significantly higher number of L. orbonalis eggs (> 92.0) compared to other species. The per cent parasitism recorded for T. evanescens was 25.3 to 2488 times and for T. chilonis, per cent parasitism was 23.5 to 2455 times higher compared to other four species tested, which indicated usefulness of these two species. In an earlier studies in the Philippines, Alpuerto (1984) reported that The results were showing more parasitization when compared with the study reported by Alpuerto (1984) from Philippines that parasitism by T. chilonis ranged from 34.6 to 51.3 per cent on L. orbonalis eggs and Nafus and Schreiner (1986) were of the opinion that T. chilonis is the most important natural enemy attacking the pyralid pests. Trichogramma viability is a feature closely related to the parasitoid host resemblance (Bezerra and Parra, 2004). In Trichogramma production, quality control considers practicality as acceptable when the rate of borne adults exceeds 85 per cent (Navarro, 1998). Thus, the species used in the present study expressed low or high affinity to the host eggs based on parasitism obtained, which is result of host acceptance. Gonçalves et al., (2003), while assessing the quality of T. pretiosum reared in Sitotroga cerealella eggs, observed viability levels above 89 per cent, which represents host acceptance and species affinity.
The per cent parasitism in the present study was similar in the two Trichogramma species, with values above 92.0 per cent. Considering that the quality control on Trichogramma production requires parasitism of more than 90 per cent (Navarro, 1998), the two species meet the criterion. It is attributed that host adaption plays significant role in acceptance of host eggs, thereby selection of promising species (Pereira et al., 2004), however, in the present study L. orbonalis eggs were offered to all species without any adaptation, thus in the present experiment on selection resulted in identifying two species, viz., T. evanescens and T. chilonis as most apt for further evaluation against L. orbonalis.

Screening of trichogrammatids at different dosages against brinjal shoot and fruit borer, L. orbonalis eggs under caged condition
From screening experiment, two species of trichogrammatids, viz., T. chilonis and T. evanescens were selected, based on their aptness for parasitizing L. orbonalis eggs, to work out dosages in a 1 sq. m cage and three dosages tried were @ 50000, 100000 and 150000 parasitoids per hectare against L. orbonalis for field evaluation.
The results revealed that there is a significant difference between the dosages used and the species selected with respect to per cent parasitism. The highest per cent parasitism, i.e., 74.1 per cent was recorded in the dosage @ 150000 adults/ha) and lowest of 44.2 percent was recorded, where dosage @ 50000 adults/ha was used. Between the two species the per cent parasitism was higher in T. chilonis compared to T. evanescens, i.e., 68.0 and 75.6 per cent, and 55.9 and 72.6 per cent in dosages @ 100000 and 150000/ha, respectively. In earlier field studies, Sangappa (1999) opined that release of T. chilonis @ 2.0 and 1.5 lakh per ha starting from 30 days after transplanting at an interval of 20 days were found effective in reducing the L. orbonalis incidence at shoot and fruit bearing stage of the crop and recorded 152.05 q per ha and 144.5 q per ha healthy fruit yield, respectively, while in other studies, the dosages of T. chilonis @ 5,00,000 adults per ha was reported effective in suppressing the pest and also recorded higher increase in yield (57 % over control) (Anon., 2001).
The present studies resulted in not only identification of most suitable species, i.e., T. chilonis, but also an effective dosages @ 100000/ha for obtaining higher suppression of the pest on brinjal.
In conclusion, six species of Trichogramma were used for selection of most suitable species, the per cent parasitism ranged between 3.60 to 93.20 per cent and the highest per cent parasitism was observed in T. evanescens (93.20 %), which was on par with T. chilonis 92.00 per cent. In a dosages experiment, the highest per cent parasitism of 74.1 per cent was recorded in the dosage having 15 adults/sq. m (150000 adults/ha) and lowest parasitism of 44.2 per cent was recorded in dosages of 5 adults/sq. m (50000 adults/ha). Among the two species highest parasitism was recorded in the T. chilonis (68.0 %) compared to T. evanescens (55.9 %) and 100,000 was found to be optimum dosages.