1025-B3
Murugan K, Vanithakumari G. and Vasugi C. 1
Studies have been conducted to assess the level of tannins in the host plants (Tectona grandis and mellingtonia hortensis) of teak defoliator, Hyblaea puera and its influence on the neem seed kernel extract (NSKE) and bacterial pesticides, Bacillus thuringiensis kurstaki (Btk). Investigation also have been done on the efficacy of neem seed kernel extract and B. thuringiensis on the mortality of teak defoliator. Higher mortality of 4 th instar larvae was evident after the treatment of neem seed kernel extract and Btk on the Mellingtonia reared larvae than the teak leaves reared insects. Higher tannin content was evident in the leaves of teak and teak leaves reared larvae. Tannin level was comparatively lower in the Mellingtonia and its reared insects. Increased tannin content in the teak leaves reared insects suggests the sequestration of tannins from the host plant. The susceptibility of larval instar fed on mellingtonia against the bio-pesticides, neem extracts and B. thuringiensis is suggestive of protective role of host plant tannins. Hence, lower mortality of larval instar of H. Puera on Mellingtonia hortensis reared leaves might have been due to lesser tannin from the host plants and their susceptibility to biological pesticides such as neem seed kernel extract and Bt toxins. Results are correlated and discussed on the possible role of plant allelochemicals on bio-pesticides for the management of forest insect pests.
Teak, a valuable timber species, is attacked by a number of insect-teak defoliator, Hyblaea puera cram. and teak skeletoniser cause major defoliation of teak in India and elsewhere (Bakasha 1990, 1993; Beeson 1941; Chaiglon 1975). The biology and ecology of H. puera was studied in detail in India (Beeson 1941; Khan and Chatterjee 1944). The reports indicate that the variation in the abundance of the pest from place to place primarily depends on the climatic conditions of the locality. The defoliator affects all parts of plants such as senescent, mature and tender leaves, which left the teak plant as bear skeleton. Our earlier studies reported that the biochemical influence of different growth phases of the host leaves of T. grandis on the feeding and growth of H. puera (Murugan and Senthil Kumar 1996) and also the effect of neem oil on the growth (Murugan 1997; Murugan et al. 1999). Murugan (2000) reported the effect of neem with microbial pesticides on teak pests. Our earlier studies also reported the adverse effect of neem alone along with other botanicals microbial on the growth, toxicity and development of other insects (Murugan et al. 1995; 1996; 1996a; 1998, 1998a; 1999, 1999a). In the present study the influence of host plant tannin in the Tectona grandis, and Mellingtonia hortensis, on efficacy of neem and Bacillus thuringiensis against. H. puera has been investigated.
Bacillus thuringiensis Berliner (B.t) is a naturally occurring, gram positive, spore-forming soil-bacterium, and preparations containing B.t. are widely used as a biological insecticide in agriculture and forestry. The entomocidal properties of B.t are mediated by Cry proteins (endotoxins) which are synthesized as parasporal crystals in the course of the sporulation of the bacterial. It is known that different types of B.t. endotoxins can damage different insect species or groups by perforating their midgut epithelium (Krieg 1986; Gills et al. 1992; Knowels 1994). Because of its host specificity, safety, and amenability to mass production, Bacillus thuringiensis has been widely exploited as a biopesticides. Neem tree has unequivocally emerged as the single most important source of insecticide. There is also now no place of skepticism regarding its potential as an effective substitute to synthetic insecticides (Schmutterer 1990).
The importance of tritrophic interactions involving pests, natural enemies and the host plants has been demonstrated repeatedly with predators and parasitoids of insects (Boethel and Eikenbary 1986, Murugan et al.2000). The literature on the interactive effects of host plants on Benz 1987, Berenbaum 1988, Barbosa 1988, Krischik 1991; Reichelderfer 1991. Depending on the system involved, pathogens can either be enhanced or inhibited by plants, plant juices or extracts, or purified secondary plant compounds. The insecticidal activity of Bacillus thuringiensis is enhanced on aspen, Populus tremuloides Michaux, foliage with higher levels of phenolic glycosides (Hwang and Lindroth 1993; Lindroth 1993; Schields 1993). Variation in insecticidal activity of Bacillus thuringiensis on oaks, Quercus spp., and aspen has been related to tannin chemistry (Schultz et al., 1993, Appel and Schultz 1994). The available data thus show that host plants can influence the activity of Bacillus thuringiensis against the insects. Because large amounts of Bacillus thuringiensis are used against host plants (Farrer et. al., 1996) can influence the gypsy moth. H. puera apart from teak as main host plant the gypsy moth, also feeds on many other plants (alternate host). However, there is lack of data on the host plant chemicals and particularly tannin on the efficacy of biological pesticides against the teak pests. Hence, this study was designed to evaluate the mortality of teak defoliator after application of neem and Bacillus thuringiensis on T. grandis and M.hortensis.
For mass culturing of insects in the laboratory, Hyblaea puera (Cramer) was collected from Tectona grandis and Mellingtonia hortensis in Bharathiar University Campus and surrounding areas of Western Ghats and cultured in cages. The newly hatched 4 th instar first instar larvae were segregated into two groups and each group was fed with T. grandis and M. hortensis ad libitum, respectively.
50 grams of neem seed kernel was powdered and dissolved in one litre of water, left to stand for 24 hr, and filtered through linen cloth to get neem seed kernel extract. It was diluted to various concentrations, Bacillus thuringiensis(Bt) and Delfin WG product that contained Bt Kurstaki, Serotype 3a, 3b, 85% and dispersing agent 15% was procured from '`Sandoz" (India) Limited, Bombay. Btk was dissolved the 100 ml of water to prepare a stock solution, from which the required concentrations ranging from 5.0, 10.0 and 15.0 _g(AI) mL were prepared. Various concentrations of neem seed kernel extract (1.0, 1.5 and 2.0%) and B. thuringiensis (5.0, 10.0 and 15.0 _g / ml) were applied evenly on T. grandis and M. hortensis leaves with a fine brush. The treated leaves were air dried. Newly moulted 4 th instar larvae of H. puera obtained from a laboratory colony were starved fro 6 h and transferred to plastic containers (6.5cm height x 5.5 cm dia.) containing treated or control T. grandis and M. hortensis leaves ( 2 larvae / container, 30 larvae / concentrations) and allowed to feed for 48hr. Fresh untreated (distilled water alone) was provided ad libitum. Mortality was recorded daily and corrected for control mortality using Abbott's formula (1925).
| Percentage mortality- | No. of dead insect ------------------------------------ x 100 No. of Insect introduced |
All the data were subjected to Duncan's Multiple Range Test (Duncan 1955).
Tannin concentration in the T. grandis and M. hortensis leaves was done by using the radial diffusion method, which utilizes a chemical reaction between tannin and protein (Hagerman 1987, 1988; Shimazaki and Miyashita 2002). Leaves of T. grandis and M. hortensis were preserved at -80 0 C and were dried at 60 0 , pulverized in a mortar. 60% aqueous acetone was added to the resulting powder and tannin was extracted. Agarose containing (0.1% bovine) serum alubumin was poured evenly into a petri dish, and three holes with 4 diameter were made on the agarose.
To determine the tannin concentration from the area of precipitation, a calibration curve for concentration versus area of precipitation was made by using purified tannin (tannin solution). The tannin extract or tannin solution was poured into each hole and the area of the precipitation was measured after 120 hr at 30 0 C. This procedure was repeated twice for each sample. The tannin concentrations of the leaves were estimated from the calibration curve.
Table 1. provides the data on morality of 4 th instar larvae of H. puera after the treatment with NSKE and Btk on T. grandis and M. hortensis. Higher mortality of larvae was evident in neem and Btk treated on M. hortensis than T. grandis.
Tannin content was high in the T. grandis leaves (5.8 mg/ml) than the M. hortensis (3.7mg/ml). Tannin content was also higher in the T. grandis fed larvae (2.9mg/ml) than the M. hortensis fed larvae (1.2mg/ml).
Table 1. Mortality of IV instar larvae of Hyblaea puera after the treatment of neem seed kernel extract and Bacillus thuringiensis on Tectona grandis and Mellingtonia hortensis
Treatment |
Mortality of NSKE and Btk on Mellingtonia hortensis |
Mortality of NSKE and Btk on Tectona grandis |
| Control NSKE (%) 1.0 1.5 2.0 Btk (µg/ml) 5.0 10.0 15.0 |
00 d 56 b 68 a 72 a 47 c 56 b 62 ab |
00 d 52 b 60 ab 64 ab 42 c 50 b 65 ab |
| Within the column means followed by the same letter(s) are not significantly different at 5% level by DMRT. |
The use of natural biochemical pesticides in commercial, agricultural and horticultural industries has increased in recent years. The bio-pesticides offer desirable alternatives to using synthetic chemicals in agricultural systems where protection of the environment and preservation of beneficial organisms are important. One such bio-pesticide of interest is insect growth regulators and azadirachtin, which metabolizes in environment (Schmutterer, 1990). Crude formulations of neem seed extracts contain limonoids that contribute to insecticidal properties. The diverse effects of neem on insect pests include feeding deterrence, reproduction disturbance and insect growth regulation among others (Mordue and Blackwell 1993). Furthermore, the compound apparently has minimal toxicity to nontarget organisms such as parasitoids, predators and pollinators (Lowery and Isman 1993) increasing acceptability for control of phytophagous insects both to pest managers and regulatory agencies. In the present study considerable mortality of larva was evident after the treatment of NSKE and Btk treatment on teak than M. hortensis. Since, crude extracts of neem seed contain limonoids that contribute to insecticidal properties (Mordue (Luntz) and Blackwell 1993). B. thuringiensis subsp. Kurstaki is one of the insecticides most commonly used to control forest insect pests (Anonymous 1994). In our earlier studies we reported use of neem alone and in combination with microbial pesticides to exert its biological effects and brought it to our mortality to various insects pests (Murugan 1997; Murugan et al. 1998 a, b; 1999; Murugan et al. 1999b).
In the present study higher mortality of larva was evident after the treatment of NSKE and Btk on teak and M. hortensis. Since, crude, extracts of neem seed contain limonoids that contribute to insecticidal properties (Mordue (Luntz) and Blackwell 1993). B. thuringiensis subsp. kurstaki is one of the insecticides most commonly used to control forest insect pests (Anonymous 1994). In the present study a strong correlation was found to exist between the tannin content in the teak and Mellingtonia and mortality of H. puera to which the neem and B. thuringiensis are applied. Higher mortality of H. puera was evident in the neem and B. thuringiensis applied on teak leaves than the NSKE and Btk treated on M. hortensis. This suggests that the higher tannin level in the teak leaves facilitate the larvae to sequester the tannin and accumulate it in the body. Hence, tannin here has a protective role over insects and helps them to resist against the biopesticides such as neem and Bt toxins. The higher mortality of larvae of H. puera fed on M. hortensis with neem and Bt toxins treatment further suggests the less availability of tannin in the host leaves and susceptible against the biopesticides.
Plant phenolic compounds have been regarded as allelochemicals against insects. Their specific mode of action is not yet clearly known. Barbehenn and Martin (1994) and Barbehenn et al. (1996) have stated that tannins can enter the haemolymph of the insect through the peritrophic envelope of the gut. Bernays and Chamberlain 1980 ; Henn 1997a; 1997b have indicated that the peritrophic envelops of insects are capable connecting to tannins by attaching to the carbohydrates (e.g. of chitin) of the envelopes (Henn 1997a;1997b). Hydrolysable tannins of oak are well known as phenolics, which can negatively influence the growth of the gypsy moth (Rossitter et al. 1988). Dunning et al. (2002) reported on the feeding behavior of the generalist migratory grasshopper, on two species of Oak with different tanning levels.
Earlier studies of Farrar et al. (1996) explained the relationship between the insecticidal activity of B. thuringiensis and the favourability of the host plant ie., those larvae feeding on Melanoplus sanguinipes variable hosts would be larger and healthier, and therefore more resistant to B. thuringiensis or that those on unfavorable hosts would be under stress, and therefore mores susceptible. They have also noted increased mortality of gypsy moth in least favored host plant (sweet gum) than in the favored host (oaks).
Some of the other studies emphasized the negative aspects of host plant tannin on the bioefficacy of insects. Extracts of foliage of a number of trees have been shown to inhibit the growth of B. thuringiensis on artificial media ( Smirnoff and Hutchinsun 1965) sweet gum was one such tree (Maksymiuk 1970). Tannins have been shown to inactivate insecticidal crystal proteins of B. thuringiensis (Luthy et al. 1985 a, b) and tannin chemistry has been implicated in variation in the susceptible host.
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