Efficacy of Microbial and IGR-based Biopesticides Against Brinjal Shoot and Fruit Borer, Leucinodes orbonalis (Guen.)

Inhaltsverzeichnis

ACKNOWLEDGEMENT

ABBREVIATIONS

ABSTRACT

LIST OF TABLES

LIST OF PLATES

LIST OG FIGURES

LIST OF APPENDICES

Chapter I INTRODUCTION

Chapter II REVIEW OF LITERATURE
2.1 General review of brinjal shoot and fruit borer
2.1.1 Syetematic position of brinjal shoot and fruit borer
2.1.2 Nomenclature of brinjal shoot and fruit borer
2.1.3 Origin and distribution of brinjal shoot and fruit borer
2.1.4 Pest status and host range
2.1.5 Status of brinjal shoot and fruit borer and its damage
2.1.6 Incidence and seasonal abundance of brinjal shoot and fruit borer
2.1.7 Life history of brinjal shoot and fruit borer
2. 2 Effects of microbial biopesticides against lepidopteran pests
2. 3 Effects of buprofezin against diffrent crop pests

Chapter III MATERIALS AND METHODS
3.1 Location and time of the study
3.2 Soil characteristics
3.3 Climate
3.4 Planting material
3.5 Design and layout of experiment
3.6 Plant cultivation
3.6.1 Land preparation for transplanting
3.6.2 Manure and fertilizer application
3.6.3 Collection and transplanting of seedlings
3.6.4 Plant spacing
3.6.5 Cultural operations
3.7 Insecticides identity
3.8 Spray schedule of treatments
3.9 Data collection
3.9.1 Data collection schedule
3.9.2 Data collection parameters and procedures
3.9.2.1 Estimation of % shoot infestation
3.9.2.2 Estimation of % fruit infestation
3.9.2.3 Estimation of the yield (t/ha) of marketable fruits
3.9.2.4 Estimation of the yield (t/ha) of infested fruits
3.10 Statistical analysis of data
3.11 Experimental methods for laboratory study
3.11.1 Mass rearing of L. orbonalis
3.11.2 Specifications of treatments
3.11.3 Treatments application methods
3.11.3.1 Topical application
3.11.3.2 Potato-dip method
3.11.3.3 Combination (topical + potato-dip) method
3.12 Data Collection
3.13 Statistical analysis

Chapter IV RESULTS AND DISCUSSION
4.1 Effectiveness of selected biopesticides on percent shoot infestation
4.2 Effectiveness of selected biopesticides on percent fruit infestation
4.3 Effects of selected biopesticides on marketable fruit yield (t/ha)
4.4 Effects of selected biopesticides on infested fruit yield (t/ha)
4.5 Comparative efficacy of different doses of buprofezin on the mortality, weight reduction and cuticular deformation of brinjal shoot and fruit borer larvae
4.5.1 Efficacy of different doses of buprofezin on the mortality and weight reduction of L. orbonalis larvae through topical application method
4.5.1.1 Effects on larval mortality
4.5.1.2 Effects on larval weight
4.5.2 Efficacy of different doses of buprofezin on the mortality and weight reduction of L. orbonalis larvae throug potato-dip method
4.5.2.1 Effects on larval mortality
4.5.2.2 Effects on larval weight
4.5.3 Comparative efficacy of different doses of buprofezin on the mortality and weight and reduction of L. orbonalis through combination (topical + potato-dip) method
4.5.3.1 Effects on larval mortality
4.5.3.2 Effects on larval weight
4.6 Effects on cuticular deformations

Chapter V SUMMARY AND CONCLUSION

REFERENCES

APPENDICES

LIST OF TABLES

1. Specification of the treatments

2. Mean percentage of shoot infestation caused by L. orbonalis at different sprayings against different treatments

3. Mean percentage of fruit infestation by L. orbonalis at different pickings against different treatments

4. Yield of marketable brinjal fruits after application of different treatments

5. Yield of infested brinjal fruits after application of different treatments

6. Mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

7. Weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

8. Mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through potato-dip method

9. Weight of L. orbonalis larvae at different time interval after treating with with different concentrations of buprofezin through potato-dip method

10. Mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

11. Weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

LIST OF PLATES

1. An infested brinjal shoot with larva

2. Infested brinjal fruit with larva

3. Life history of brinjal shoot and fruit borer

4. Experimental field with brinjal plants at seedling stage

5. Brinjal plants in the field during treatment application

6. Steps followed for mass rearing of Leucinodes orbonalis

7. Representative photographs (A-C) showing consecutive steps for topical application method

8. Representative photographs (A-C) showing the consecutive steps for potato-dip method

9. Representative photographs (A-D) showing the successive steps for combination method

10. A healthy brinjal plant with fruits

11. An infested brinjal shoot

12. Some infested brinjal fruits after picking

13. An infested brinjal fruit with larvae inside

14. Experiment in the laboratory

15. Representative photomicrographs of the larvae of Leucinodes orbonalis after 7 days of treatment application

LIST OF FIGURES

1. Allocation of treatments in the experimental plots

2. Structural formula of emamectin benzoate

3. Structural formula of abamectin

4. Structural formula of spinosad

5. Structural formula of buprofezin

6. Percent protection of brinjal shoots over control

7. Percent fruit protection over control

8. Increase in percent marketable fruit yield over control

9. Percent weight reduction of L. orbonalis larvae over control

LIST OF APPENDICES

I. Analysis of variance for mean percentage of shoot infestation caused by L. orbonalis at different sprayings against different treatments

II. Analysis of variance for mean percentage of fruit infestation by L. orbonalis at different pickings against different treatments

III. Analysis of variance for the yield of marketable brinjal fruits after application of different treatments

IV. Analysis of variance for the yield of infested brinjal fruits after application of different treatments

V. Analysis of variance for mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

VI. Analysis of variance for mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through potato-dip method

VII. Analysis of variance for mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

VIII. Analysis of variance for mean weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

IX. Analysis of variance for mean weight of L. orbonalis larvae at different time interval after treating with with different concentrations of buprofezin through potato-dip method

X. Analysis of variance for mean weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

XI. Pattern of mean monthly rainfall, humidity, and temperature during the experimental period (January to June, 2014)

ACKNOWLEDGEMENT

All the praises, gratitude and thanks are due to almighty Allah (subbhanahu-wa-taalah) who enabled to complete this thesis work successfully for the degree of Master of Science (MS) in Entomology.

The author feels proud to express his deepest appreciation, heartfelt gratitude and indebtedness to his research supervisor, Dr. Gopal Das, Associate Professor, Department of Entomology, Bangladesh Agricultural University (BAU), Mymensingh for his valuable guidance, constructive criticism, encouragement and suggestions during the experimental period and completion of this thesis.

The author is highly grateful and feels proud to express his honour to the co-supervisor Professor Dr. Mohammad Mahir Uddin, Department of Entomology, Bangladesh Agricultural University, Mymensingh for the valuable guidance and continuous inspiration in the preparation of this thesis.

The author expresses his cordial thanks to all reverend teachers and staffs of the Department of Entomology, Bangladesh Agricultural University, Mymensingh. The author gratefully acknowledges the cordial cooperation, friendly collaboration, fruitful advice and help received from many persons from the starting to the end of this thesis.

The author owes a debt of gratitude to his beloved parents, sisters, brothers and other family members for their blessing, inspiration, sacrifice and moral support throughout the entire period of his academic life.

The author

ABBREVIATIONS

EFFICACY OF MICROBIAL AND IGR-BASED BIOPESTICIDES AGAINST BRINJAL SHOOT AND FRUIT BORER, Leucinodes orbonalis (Guen.)

In the present study, efficacy of some microbial and IGR-based biopesticides against the infestation of brinjal shoot and fruit borer (BSFB), Leucinodes orbonalis (Guen) was evaluated in the Entomology Field Laboratory, Bangladesh Agricultural University (BAU), Mymensingh during January to July 2014. Biopesticides were applied either individually or in some selected combinations and their efficacy was evaluated on different parameters viz. percent shoot infestation, percent fruit infestation, infested and marketable fruit yield (t/ha). It was found that all the treatments significantly reduced percent shoot and fruit infestation and significantly increased marketable fruit yield. The best result was found in case of combined treatment buprofezin + emamectin benzoate treated plots (70.75% shoot and 63.99% fruit protection over control; highest marketable fruit yield of 9.94 t/ha) whereas the least protection was obtained from buprofezin (1 ml/L) treated plots (17.87% shoot and 15.66% fruit protection over control; lowest marketable fruit yield of 6.05 t/ha). In the laboratory, different concentrations of buprofezin was applied through topical, potato-dip and combined (topical + potato-dip) methods on the 2nd instar larvae of BSFB and then mortality, weight reduction or cuticular changes were observed at 3 HAT (hours after treatment), 3 and 7 DAT (days after treatment). No mortality or weight reduction was observed at 3 HAT. The significant level of mortality and weight reduction was observed at 3 DAT in all applying methods but the maximum mortality and weight reduction was found at 7 DAT. It was also observed that potato-dip method was highly effective than topical method regarding mortality and weight reduction. Cuticular abnormalities were found when larvae were treated with higher concentrations of buprofezin in comparison with that in the water-treated control. Moreover, larval mortality and weight reduction were clearly dose-dependent.

CHAPTER I INTRODUCTION

Brinjal (Solanum melongena Linn.) is one of the most popular and year round vegetable crop cultivated widely throughout Bangladesh and covers about 15% of the total vegetable cultivation area of the country (Rahman, 2005). It is usually grown as a seasonal crop and widely cultivated both in rabi and kharif seasons. In Bangladesh it is the second most important vegetable crop after potato in relation to its total production. Asia has the largest brinjal production which comprises about 90% of the total production area and 87% of the world production (FAO, 2000). Brinjal belong to the family Solanaceae and is normally a self-pollinated annual crop. Due to its nutritive value, consisting of minerals like iron, phosphorous, calcium and vitamins like A, B and C, unripe fruits are used primarily as vegetable in the country. It is also used as a raw material in pickle making and as an excellent remedy for those suffering from liver complaints. It is used in Ayurvedic medicine for curing the diabetes. It is also used as a good appetizer. It is a good aphrodisiac, cardiotonic, laxative and reliever of inflammation.

Brinjal is susceptible to attack of various insects from seedling to fruiting stage. This crop is infested by 53 different insect species (Nayer et al., 1995) including (1) Brinjal shoot and fruit borer, BSFB (Leucinodes orbonalis Guen.) (2) Epilachna beetle (Epilachna vigintioctopunctata Fab.), (3) Aphid (Aphis gossypii Glover), (4) Jassid (Amrasca biguttula Ishida), (5) White fly (Bemisia tabaci Gennadius) and so on. In Bangladesh about eight insect species are considered as major pests causing damage to the crop (Biswas et al., 1992). Among these insect pests brinjal shoot and fruit borer, Leucinodes orbonalis Guen. (Lepidoptera: Pyralidae) is the most destructive pest and is considered to be the limiting factor in quantitative as well as qualitative harvest of brinjal fruits. In early stage of the crop growth, larva bores into the shoots resulting in drooping, withering and drying of the affected shoots. During the reproductive stage, tiny larva bores into the flower buds and fruits, the bored holes are invariably plugged with excreta. The infested fruits become unfit for human consumption due to loss of quality and lose their market value. It is also reported that there will be reduction in vitamin C content to an extent of 68 percent in the infested fruits (Hemi, 1955). Damaged fruits are identified by the presence of exit holes. BSFB larvae attack shoots in early stage affecting the plant vascular system whereas in later stages larvae prefer fruits. Only the larvae of this pest cause 12-16% damage to shoots and 20-60% to fruits (Alam, 1969). The pest is very active during the rainy and summer season and often causes more than 90% damage (kallo, 1988). The yield loss has been estimated up to 86% in Bangladesh (Ali et al., 1980) and up to 95% in India (Nasr et al., 2010). This pest can also infest potato and other solanaceous crops and wild plants (Karim and Islam, 1994).

Chemical control is the most common practice in Bangladesh to control L. orbonalis as well as to produce blemish-free brinjal fruits. More accurately, 98% brinjal-growers in Jessore district (southern part of Bangladesh) relied exclusively on the use of conventional pesticides and they use those pesticides 140 times or more in the 6-7 month cropping season (AVRDC, 2003). According to Pesticide Association of Bangladesh (1999), use of pesticide on growing brinjal was 1.41 kg/ha whereas it was overall 1.12 kg for vegetables and only 0.20 kg/ha in rice. Brinjal being a vegetable crop, use of chemical insecticides will leave considerable toxic residues on the fruits which is certainly harmful for human health. In addition, sole dependence on chemical insecticides for controlling L. orbonalis has lead to insecticide resistance, secondary pest outbreak, killing of non-target organisms, environmental hazards etc. The frequent use of insecticide is ecologically unsafe and economically unviable too. Insecticides may provide quick solution to the problem but their use must be judicious and other control approaches should be given emphasis in managing the BSFB. Considering the above facts a research work has been undertaken to investigate the efficacy of some biopesticides against BSFB.

The term biopesticide is rapidly gaining popularity in the current climate of environmental awareness and public concern. This term is derived from two words, biological and pesticide, referring to pesticides of natural or microbial origin that have limited or no adverse effects on the environment or beneficial organisms. The Environmental Protection Agency (EPA) considers biopesticides to have different modes of action than conventional or traditional pesticides, with greater selectivity and considerably lower risks to human, wildlife and the environment. Biopesticides may be derived from a variety of sources, including bacteria, viruses, fungi and protozoa, as well as chemical analogues of naturally occurring biochemicals such as pheromones and insect growth regulators (IGRs). They are considered as third-generation pesticides that are environmentally sound and closely resemble or are identical to chemicals produced by insects and plants.

Among the microbial insecticides, spinosad, emamectin benzoate and abamectin (Kumar and Devappa, 2006) are the most promising insect control agents. The role of microbial insecticides, in lepidopteran insect pest management has obvious advantages in terms of effectiveness, specificity and safety to nontarget organisms and other components related to biosphere. Therefore, these microbial insecticides were evaluated against BSFB under the field condition in the present study. Moreover, there are some natural and synthetic analogs which are capable of interfering with the processes of growth, development, moulting and metamorphosis of the target pests. These chemicals have been called “Insect Growth Regulators” (IGRs) and they are very potential birational pesticides.

The mode of action of IGRs is not central nervous system-oriented but kill insects potentially through cessation of moulting process (Asai et al., 1985). Buprofezin, a potent chitin synthesis inhibitor (CSI) reduces pest population by preventing moulting through inhibition of chitin bio-synthesis. Moreover, buprofezin has multiple effects on the target pests like reduction of fecundity, egg hatchability, egg sterility, production of abnormal larvae and pupae (Ragaei and Sabry, 2011). Buprofezin was found to be very effective against hemipteran pests, few lepidopteran larvae, spiders etc (Deng et al., 2008; Izawa et al., 1985 and Nagata, 1986). It was reported that buprofezin is an ecofriendly biopesticide that is safe for non-target organisms, highly biodegradable and action is target pest specific (Sontakke et al., 2013). However, very little information is available yet on the impact of buprofezin against brinjal shoot and fruit borer, L. orbonalis. Hence, the experiment was planned to evaluate the efficacy of buprofezin against BSFB in the field as well as to observe its effect on the mortality, weight reduction and cuticular deformations of BSFB larvae under laboratory conditions. The present study was designed to fulfill the following objectives:

I. to evaluate individual effect of different microbial and IGR-based biopesticides against BSFB infestation in the field
II. to evaluate the combined effects of some selected biopesticides against BSFB infestation
III. to identify the most effective treatment or treatment combinations based on target action
IV. to find out the efficacy of selected biopesticides on the marketable versus infested yield (t/ha)
V. to investigate the effect of buprofezin (IGR) on the mortality, weight reduction and cuticular deformations of BSFB larvae under laboratory conditions

CHAPTER II REVIEW OF LITERATURE

Brinjal (Solanum melongena Linn.) is the most common, delicious, popular and one of the principal vegetable in Bangladesh and other parts of the world (Nonnecke, 1989). A wide range of insect pests attack this crop due to cultivation throughout the year, out of these brinjal shoot and fruit borer (BSFB), Leucinodes orbonalis (Guen.) is considered as most serious pest occurring either sporadically or as outbreak every year wherever the crop is grown affecting quality and quantity of brinjal adversely. It damages the tender shoots and fruits. The yield loss caused by this pest may up to 85%. Control of BSFB is difficult as it is an internal feeder and farmers usually rely on different toxic insecticides for controlling this destructive pest. However, the use of biopesticides for managing this pest is getting popularity day by day due to their specificity to the target pest, safety to natural enemies and reduced risks for human health and environment. Literature cited below under the following headings and subheadings reveal some information about this study.

2.1 General review of brinjal shoot and fruit borer, Leucinodes orbonalis

2.1.1 Systematic position of brinjal shoot and fruit borer

Phylum: Arthropoda

Class: Insecta

Sub-Class: Pterygota

Division: Endopterygota Order: Lepidoptera

Family: Pyralidae

Genus : Leucinodes

Species: Leucinodes orbonalis

2.1.2 Nomenclature of brinjal shoot and fruit borer

Brinjal shoot and fruit borer (Leucinodes orbonalis) is one of the most destructive insect pests of brinjal (Solanum melongena L.) in Bangladesh. It is phytophagous in nature and belongs to the order Lepidoptera and family Pyralidae. The genus Leucinodes has three species, Leucinodes orbonalis Guen., Leucinodes diaphana Hamps. and Leucinodes apicalis Hamps. ( Alam, 1969).

2.1.3 Origin and distribution of brinjal shoot and fruit borer

Hampson (1986) first described L. orbonalis. Accoding to Butani and Jotwani (1984) L. orbonalis, the most serious pest of brinjal is not only distributed in the Indian sub continent but also in South Africa, Congo and Malaysia. Brinjal plants are severely attacked by shoot and fruit borer in the tropics but not in the temperate zone (Yamaguchi, 1983).

2.1.4 Pest status and host range

L. orbonalis is the serious pest of brinjal and it is also reported to infest potato and other solanaceous crops . Several wild species of solanum are also attacked by this pest (Karim and Islam, 1994). The larvae also feed on pods of green peas (Alam et al . , 1964). Ishaque and Chaudhuri (1983) observed that besides brinjal, some other plants served as host plant and caused varying levels of infestation during different periods of the year. The plants Solanum nigrum, S. indicum, S. torvum, S. myriacanthum, S. tuberosum have been reported as alternative host of the pest.

2.1.5 Status of brinjal shoot and fruit borer and its damage

Brinjal shoot and fruit borer is very injurious to brinjal during the rainy and summer seasons. The losses incurred due to its infestations are sometimes reported to be more than 90% (Kalloo, 1988).

The damage caused by L. orbonalis starts soon after transplanting of seedling and continues till the harvest of the fruits. The caterpillar after hatching begins to search for soft and tender shoots to bore. It is active from the beginning of its life. In young plants, the larvae bore into the petioles and midribs of large leaves and young shoots. After entering into the host the larvae close the entry holes with their excreta and feed inside by its mandibles (Butani and Jotwani, 1984). The infested shoots drop off due to disruption of vascular system and ultimately withered (Alam and Sana, 1962) and the borers continue boring the stem until it encases itself.

At a late stage of plant growth, when the flower buds comes out the larvae at first bore generally though the calyx and later into the flower buds and the fruits without leaving any visible sign of infestation and feed insides (Butani and Jotwani, 1984). The infested flower buds dry and shed. Infested fruits show exit hole along with excreta. The caterpillar rests in a cell of fruit. The affected fruit, when cut open is sometimes found rotting with full of dark excreta. Moulds grow therein and thus make the fruit unfit for human consumption and marketing.

The full grown larva comes out through the hole of the fruits and drop on the ground for pupation in the soil or plant debris. The percent infestation of fruits is more than that of the shoot (Alam and Sana, 1962). The fruit infestation may even reach above 60% during the rainy season in Bangladesh.

2.1.6 Incidence and seasonal abundance of brinjal shoot and fruit borer

The seasonal history of shoot and fruit borer varies considerably due to different climatic conditions throughout the year. Hibernation does not take place and the insects are found active in summer months, especially in rainy season. A study revealed that the population of L. orbonalis began to increase from the first week of July and peaked (50 larvae per 2 sq m) during the third week of August. The population of this pest was positively correlated with average temperature, mean relative humidity and total rainfall (Shukla, 1989).

Brinjal shoot and fruit borer is less active during February to April (Alam, 1969). During winter months, different stages of this pest last for longer periods and overlapping generations were observed. There is a considerable mortality of larvae caused by predatory black ant, Camponotus compresses during summer (Alam, 1969). Pupal mortality has observed during rainy season due to attack of ichneumonid parasitoid and the wasp, Trichogramma chilonis (Hagerman, 1990). The black ant, Camponolus compressus also attacks the adult moths. Maximum population of adult moths has been observed during the months of December and April (Alam, 1969).

Panda (1999) conducted a field experiment on 174 brinjal cultivars for resistance to L. orbonalis at Bhubaneswar, India. None of brinjal entries was immune to larval attack of shoots and fruits. The mean performance of shoot infestation varied from 1.61 to 44.11 % and fruit damage varied from 8.5 to 100.0%. Maximum shoot damage was recorded at 75 DAT and 99, 114 DAT in susceptible and resistant cultivars, respectively.

Pawar et al. (1986) reported that infestation of shoots began 30 days after transplanting, peaked in the 2nd week of September and reached zero in the 1st week of November. Fruit was infested from the 3rd week of September and the infestation peaked in the 2nd week of January. On the summer crop, shoots were infested from 3rd week of January and the infestation peaked in the 2nd week of February. Infestation of fruit peaked in the 1st week of April. Infestation levels were lower during winter than during summer season.

Patel et al. (1988) showed that brinjal (cv. Doll -5) transplanted in May was more damaged by L. orbonalis than the crops transplanted in January, July, September and November. They also observed that among environmental factors, low variation in minimum and maximum temperatures, high relative humidity and suitable rainfall enhanced the population of this pest.

Butani and Jotwani (1984) reported that the brinjal shoot and fruit borer causes 1 to 16% damage to shoots and 16 to 64% to fruits in Bangladesh. Ali et al. (1980) made a brief observation on the incidence of shoot and fruit borer on 12 cultivars of brinjal. They observed that the cultivar, Baromashi showed no shoot and fruit infestation. The lowest percentage of fruit infestation (25%) occurred in Singnath and the highest (86%) in Jhumki.

Alam et al. (1964) observed that the damage caused by the brinjal shoot and fruit borer varies from 12-16% in shoots and 20-63% in fruits.

Alam and Sana (1962) reported that the percent infestation of fruits is more than that of shoots. The fruit infestation may even reach above 60% during the rainy season in Bangladesh.

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Plate 1. An infested brinjal shoot with larva

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Plate 2. Infested brinjal fruit with larva

2.1.7 Life history of brinjal shoot and fruit borer Adult

Moths come out of pupal cocoons at night. Young adults are generally found on the lower leaf surfaces following emergence. Females are slightly bigger than males. The abdomen of the female moth tends to be pointed and curl upwards, whereas the male moth possesses a blunt abdomen. The moth is white but has pale brown or black spots on the dorsum of thorax and abdomen. Wings are white with a pinkish or bluish tinge and are ringed with small hairs along the apical and anal margins. The forewings are ornamented with a number of black, pale, and light brown spots. The moth measures 20 to 22 mm across the spread of wings. Longevity of adults was 1.5 to 2.4 days for males and 2.0 to 3.9 days for females. The pre-oviposition and oviposition periods were 1.2 to 2.1 and 1.4 to 2.9 days, respectively (Mehto et al., 1983). Mehto et al. (1983) reported that in India the egg, larval and pupal periods were 5.4, 17.5 and 9.8 days, respectively; the lifespan of adult males and females was 1.5-2.4 and 2.0-3.9 days, respectively. The number of eggs produced per female ranged from 84.5 in January to 253.5 in May.

Larvae are commonly known as caterpillars. The newly hatched larvae are about 1.5 mm long and dull- white in color, whereas the full grown larvae are 15 to 18 mm long, light pink in color and have prominent tubercles with hair on each thoracic and abdominal segments. Larvae go through at least five instars (Atwal, 1976) and there are reports of the existence of six larval instars. Larval period lasts 12 to 15 days in the summer and up to 22 days in the winter (Kumar and Johnsen, 2000). The full grown larvae showed a prepupal period of 3-4 days before Pupation.

The full-grown larvae comes out from the infested shoot and fruit for pupation on the stems, dried leaves and shoot or debris on the soil surface under the plants (Alam and Sana, 1962). The pupa is formed within a boat-shaped cocoon of dirty brown silk, which is spun, by the full-grown larvae before pupation. The color and texture of the cocoon matches the surroundings making it difficult to detect. Some studies indicate the presence of cocoons at soil depths of 1 to 3 cm. The anal segment of the male pupa is devoid of bristles. Whereas the female pupa has eight bristles with incurred tips at the anal segment ( Alam and Sana, 1962). The incubation, larval and pupal period are 3 to 5, 12 to 15 and 7 to 10 days during the summer and 7 to 8, 14 to 22 and 13 to 15 days in the winter, respectively (Alam and Sana, 1962; Butani and Jotwani, 1984). The life cycle is completed in 34 to 60 days with five to more generations per year (Alam and Sana, 1962; Alam, 1969).

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Plate 3. Life history of brinjal shoot and fruit borer

2.2. Effects of microbial biopesticides against lepidopteran pests

Patil et al. (1999) reported that, spinosad 48 SC at 100 g a.i/ha recorded minimum cotton bollworm incidence of 8.50 percent which was on par with its lower dosage 75 g a.i/ha. These two doses were significantly superior to the other dosages and also with standard check insecticide treatments except cypermethrin 75 g a.i/ha, which recorded minimum bollworm incidence of 6.11 percent and was on par with spinosad 48 SC @ 100 g a.i/ha. Maximum incidence was recorded in untreated control (41.12%).

Spinosad and emamectin benzoate have given good control of the southern armyworm (Schuster, 2001) and the tomato pinworm (Stansly and Connor, 1998).

Dandule et al. (2000) studied the efficacy of spinosad 48 SC against cotton bollworm in comparison with some synthetic pyrethroids. Spinosad 48 SC @ 50 and 75 g a.i/ha was effective as that of synthetic pyrethroids in controlling the bollworms.

John et al. (2000) evaluated the commercial formulation spinosad 2.5 SC against cabbage pests at four different concentrations (10, 15, 20 and 25 g a.i/ha) with cypermethrin (60 g a.i/ha), quinalphos (250 g a.i/ha) and Bt biobit (32 g a.i/ha). Results indicated that spinosad 2.5 SC was better in controlling cabbage head borer, Hellula undalis (Fab.) when applied at 15, 20 and 25 g a.i/ha.

Swamy et al. (2000) reported that spinosad 45 SC (25.33%) was equally promising for the control of pink boll worm as the case with commonly used quinalphos (26.35%) and cypermethrin (27.18%).

Banerjee et al. (2000) reported that spinosad 48 SC @ 50 and 75 g a.i/ha was found to be the best treatment and proved to be effective in reducing green boll damage and open boll damage thus contributing to a higher yield of seed cotton.

Dey and Somchoudhary (2001) reported that the spinosad 48 SC (Spinosyn A and D) against three important lepidopteran pests of cabbage viz., diamondback moth, Plutella xylostella (Lin.); cabbage head borer, H. undulis and leaf caterpillar, Sporoptera littoralis (Bios.). Spinosad at a dose level of 15-25 and 20-25 g a.i/ha found to be effective against all the three lepidopteran pests.

Walunj et al. (2001) revealed the superiority of spinosad 48 SC @ 15 g a.i/ha against diamondback moth for a period of one week with better yield of marketable heads. None of the insecticides showed any phytotoxic effect on cabbage crop.

Ghosh et al. (2001) reported that synthetic pesticide avermectin (0.01%) was found to be effective against pest complex of cabbage.

Ashok et al. (2001) studied the effect of some novel insecticides against P. xylostella. Results showed that novaluron 0.00075 percent was found to be most effective followed by abamectin 0.00045 percent and diafenthiuron 0.12 percent.

Gowda et al. (2003) reported that spinosad 45 SC at higher dosage (50 g a.i/ha) recorded significantly lower pod damage and higher grain yield compared to endosulfan @ 700 g a.i/ha in pigeon pea.

Different microbials B. thuringiensis Var. Kurstaki (Btk, dipel 8L), Sacharopolyspora spinosa (spinosad 45 SC, tracer) and B. bassiana were tested alone and in combinations against third instars larvae of H. armigera. The individual treatments of S. spinosa (0.0018 or 0.003%) and its combinations (0.006 or 0.0013%) with Btk (0.02 to 0.08%) resulted in significantly higher larval mortalities of 100 percent and 64.8 to 110.00 percent, respectively (Sridevi et al., 2004).

Puranik et al. (2002) evaluated different B. thuringiensis (Bt) formulations in comparison with neem and chemical insecticides against brinjal shoot and fruit borer. Among the different treatments, five sprays of dipel 8L @ 0.2 percent at 10 days interval resulted in

minimum shoot (9.56%) as well as fruit (11.78%) infestation and maximum yield of marketable fruits (196.96 q/ha) and proved to be the most effective treatment.

Yin (1993) reported that spraying of Bt emulsion against shoot and fruit borer in brinjal resulted in 78.8-100 percent control over untreated check.

Murali et al. (2002) reported that the phorate application at transplanting followed by foliar spray of Bt + carbaryl reduced the shoot infestation ranging from 6.71 to 9.30 percent. Fruit infestation on number basis was minimum in the treatment of phorate application at transplanting and followed by combined application of Bt + endosulfan and Bt + carbaryl recording 8.71 to 9.83 percent.

Kanna et al. (2005) evaluated emamectin benzoate 5 SG against tomato fruit borer, H. armigera. The insecticide formulation at 10 g a.i/ha and 8.75 g a.i/ha was effective against the fruit borer when compared to profenofos 50 EC (750 g a.i/ha).

Aparna and Dethe (2005-2006) conducted an experiment on bioefficacy study of biorational insecticide on brinjal. Field experiments were undertaken for two cropping seasons during kharif 2005 and summer 2006. From the study it was found that spinosad afforded moderate control of jassid, whitefly and aphid. However, it was found to be the most effective against BSFB. The lowest percent fruit infestation of 13.34 and 13.69 and 7.89 and 8.21 percent on number and weight basis in kharif and summer season respectively was found in spinosad 72 gm a.i/ha treated plots. Spinosad at 72 g a.i/ha resulted in notable yield of healthy brinjal fruits. The marked increase in healthy fruit yield resulting due to lowest fruit damage was recorded in spinosad 72 gm a.i/ha treatment where the maximum marketable brinjal fruit yield of 20.41 t/ha was recorded.

Udikeri et al. (2004) reported that emamectin benzoate 5 SG at 11 g a.i/ha resulted in significantly the lowest larval population (0.10/plant) of cotton bollworm and was found at par with spinosad 48 SC @ 50 g a.i/ha (0.14/plant) and indoxacarb 15 SC 75 g a.i/ha (0.16/plant). The damage due to bollworm was the least in emamectin benzoate (4.19%), which resulted in significantly more number of good opened bolls and less number of bad opened bolls along with the highest seed cotton yield (15.93 q/ha).

Bhemanna et al. (2005) evaluated emamectin benzoate (proclaim 5% SG), a new insecticide against okra fruit borer. Emamectin benzoate @ 8.50 g a.i/ha recorded lower fruit borer damage and higher fruit yield and was highly promising against okra fruit borer complex.

Vishal and Ujagir (2005) evaluated newer molecule spinosad (tracer) 45% SC along with other insecticides. Among different treatments lower number of H. armigera, Maruca vitrata (Geyer) and Melanagromyza obtusa (Malloch) larvae were recorded in spinosad 90 g/ha and spinosad 73 g/ha and also recorded lower pod damage compared to other treatments.

Kumar and Devappa (2006) reported that when emamectin benzoate was tested against brinjal shoot and fruit borer during 2002-03 and 2003-04. The results indicated that the application of proclaim 5 SG (emamectin benzoate) @ 200 g/ha was found effective in reducing the dead hearts and also fruit damage in brinjal. The total yield was also higher in this treatment.

Murugaraj et al. (2006) reported that emamectin benzoate (proclaim 5 SG) @ 11 g a.i/ha was highly effective in reducing the larval population of H. armigera, fruit damage as well as in increasing the tomato yield.

Anil and Sharma (2010) studied on the efficacy of spinosad and emamectin benzoate. They found that spinosad and emamectin benzoate were effective in suppressing the fruit infestation by BSFB.

2.2.2 Effects of buprofezin (IGR) against different crop pests

Buprofezin is an insect growth regulator (IGR) that disrupts the development of immature forms of insects by interference with chitin synthesis and it is mostly effective against homopterans pests such as planthoppers, leafhoppers, and whiteflies on rice. Unlike traditional chemical insecticides, buprofezin reduces pest population by preventing moulting through the inhibition of chitin bio-synthesis. Chitin is a major component of the insect exoskeleton. Insects poisoned with buprofezin are unable to synthesize new cuticle, thereby preventing them from moulting successfully to the next stage and ultimately leading to death by fracturing the cuticle. Because this insecticide has generally been considered to have good efficacy against the target pests while being harmless to beneficial insects, it has been used widely in integrated pest management (IPM) programs (James, 2004).

Research proved that buprofezin affected egg production and hatching in several coccinellids ( Magagula and Samways, 2000) as well as in hemipterans (Smith, 1995).

Numerous studies proved that buprofezin has significant effects on the physiology of molting and the feeding behavior of economically injurious insects (Asai et al., 1985; Gu et al., 1993; Heong, 1988).

Smith (1995) showed that buprofezin caused significant larval mortality and reduced egg production in the scale-feeding coccinellid, Chilocorus circumdatus Gyllenhal.

Valle et al. (2002) considered a chitin synthesis inhibitor against larvae of lepidoptera because it interferes with chitin formation by blocking the polymerizations process of N- acetyl glucose amine units (Ishaaya and Horowitz, 1998).

Nasr et al. (2010) found that buprofezin caused reasonable mortality in Spodoptera littoralis larvae.

Das (2013) found that buprofezin had no direct effect on the mortality of rice weevils regardless of the concentrations. Buprofezin at 300 ppm in rice grains significantly inhibited progeny production while lower doses (200 and 100 ppm) had no significant effect but virtually reduces progeny number.

Ragaei and Sabri (2011) found that buprofezin was very effective against the fourth instar larvae of the cotton leafworm, Spodoptera littoralis and caused significant mortality and growth reduction of the leafworm larvae.

CHAPTER III MATERIALS AND METHODS

Microbial and insect growth regulator (IGR) based biopesticides were evaluated individually and in some selected combinations against the infestation caused by brinjal shoot and fruit borer, Leucinodes orbonalis (Guen.) in field conditions. Moreover, the efficacy of buprofezin (a chitin synthesis inhibitor) on the mortality, weight reduction and cuticular deformations of L. orbonalis larvae was carried out under laboratory conditions. Methodological procedures used in the field and laboratory are briefly described in this chapter. Methodological procedures for field experiments were as follows:

3.1 Location and time of the study

The field experiments were conducted in the field laboratory of the Department of Entomology, Bangladesh Agricultural University (BAU), Mymensingh, located at 24.75"N latitude 90.5"E longitude at a mean elevation of 7.9 to 9.1 m above the sea level during January to July 2014. The effectiveness of different biopesticides for the management of brinjal shoot and fruit borer was studied in the brinjal field. Concurrently, effects of buprofezin on the mortality, growth inhibition and cuticular deformations of brinjal shoot and fruit borer larvae were studied under laboratory conditions. Details of the materials and methods used in this study are presented bellow.

3.2 Soil characteristics

The soil of the field experiment area was under Old Brahmaputra Alluvial Tract under the Agro Ecological Zone 9 with sandy loam soil and texture having good irrigation and drainage facilities. The pH of the soil was within the range of (6.3 - 7.6) with an organic matter content of 0.8 to 1.6% (Anon, 1978).

3.3 Climate

The field experiments were conducted under sub-tropical climate, which is characterized by high temperature, high humidity and heavy precipitation with occasional gusty winds in kharif season (April-September) and scanty rainfall associated with moderately low temperature during the rabi season (October-March).

3.4 Planting material

Brinjal variety, Amjhuri which is a local cultivar, was selected for the experiment.

3.5 Design and layout of experiments

The field experiments were consisted of nine treatments. All the treatments were laid out in the Randomized Complete Block Design (RCBD). Each of the treatments was replicated for three times. The whole experimental field was about 23 m in length and 9 m in width which was then divided into 3 equal blocks. Each of the blocks had 9 equal plots and finally a total of 27 plots were made in the specified area for conducting the experiments. The size of a unit plot was 2 m X 2 m. Two adjacent unit plots and blocks were separated by 60 cm and 80 cm apart, respectively. Plots were allocated randomly and they were separated in such way so that impact of every treatment can be quantified.

3.6 Plant cultivation

3.6.1 Land preparation for transplanting

The experimental land was first opened with a country plough. The land was then gradually ploughed and cross-ploughed several times with a power tiller to obtain desirable final tilth that was followed by laddering and spading .The stubbles of the crops and uprooted weeds were removed from the field and the land was properly leveled. Finally, the unit plots were prepare d as 10cm raised beds along with the addition of basal doses of manures and fertilizers.

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Figure 1. Allocation of treatments in the experimental plots

3.6.2 Manure and fertilizer application

Cowdung and other chemical fertilizers were applied as recommended by Rashid (1993) for eggplant at the rate of 15000 kg cowdung and 250, 150, 125 kg of Urea, TSP and MP respectively per hectare. The full doses of cowdung, TSP and a half of MP were applied as basal dose during land preparation. The entire dose of urea and the rest of MP were applied as top dressing. The first top dressing with one third of urea was made at 20 days after transplanting followed by second top dressing comprising one third of urea and one fourth of MP at the time of flower initiation followed by last top dressing comprising rest of urea and MP at the time of fruit initiation.

3.6.3 Collection and transplanting of seedlings

Healthy and disease free brinjal seedlings were collected from the local nursery. T he collected seedlings were transplanted in the experimental plots at the rate of 4 seedlings /plot.

3.6.4 Plant spacing

The plant spacing was followed viz. 80 cm X 60 cm.

3.6.5 Cultural operations

A light irrigation was applied just after transplanting of seedlings. To avoid stagnant of water proper irrigation was provided. Weeding and mulching were done as recommended by Rashid (1993). Damaged seedlings were replaced by new ones from the stock. Supplementary irrigation was applied at an interval of 2-3 days. Weeding was done when necessary. The MP and urea fertilizers were top dressed in 3 splits as described earlier.

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Plate 4. Experimental field with brinjal plants at seedling stage

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Plate 5. Brinjal plants in the field during treatment application

3.7 Insecticides identity

In the present study, four insecticides from different groups have been evaluated against the infestation caused by brinjal shoot and fruit borer. Their trade name, chemical name, formula, properties and mode of actions are given below in brief:

[a] Trade name: Suspend 5 SG

Chemical name: Emamectin benzoate

Empirical formula: C49H75NO13. C7H6O2 (B1a), C48H73NO13. C7H6O2 (B1b)

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Figure 2. Structural formula of emamectin benzoate

Properties: Emamectin is the 4”-deoxy- 4”-methylamino derivative of abamectin, a 16- membered macrycyclic lactone produced by the fermentation of the soil actinomycete Streptomyces avermitilis. Emamectin differs from avermectins B1a and B1b by the presence of a hydroxyl group at the 4”- epimethylamino group rather than the 4”- position. It is generally prepared at the salt with benzoic acid, emamectin benzoate, which is a white or faintly yellow powder.

Mode of action: Emamectin benzoate is basically a chloride channel activator effect causing prevention of muscle contraction, cessation of feeding and finally death. It is a local systemic insecticide rather than true systemic i.e. it has strong translaminar movement properties. It has very good contact and stomach action.

[b] Trade name: Benten 1.8 EC Chemical name: Abamectin

Empirical formula: C48H72O14 (B1a); C47H70O14 (B1b)

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Figure 3. Structural formula of abamectin

Properties: Abamectin is a mixture of avermectins containing more than 80% avermectin B1a and less than 20% avermectin B1b. It is isolated from a set of eight molecules produced by the original soil bacterium Streptomyces avermitilis. It is odorless, light yellow crystalline powder. Melting point: 169.4-161.8 ºC.

Mode of action: Abamectin act as insecticide as well as acaricide with contact or stomach actions sometimes with local systemic activity. The target for abamectin involves the gamma-aminobutyric acid (GABA) receptor in the peripheral nervous system. The compound stimulates the release of GABA from nerve endings and enhances the binding of GABA to receptor sites on the post-synaptic membrane. This enhanced GABA binding results in an increased flow of chloride ions into the cell, with consequent hyper-polarisation and elimination of signal transduction, resulting in an inhibition of neurotransmission.

[c] Trade name: Libsen 45 SC

Chemical name: Spinosad [spinosyn A (60-70%) + spinosyn D (30-40%)]

Empirical formula: C41H65NO10 (spinosyn A ); C42H67NO10 (spinosyn D)

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Figure 4. Structural formula of spinosad

Properties: Spinosad is a fermentation product of soil bacteria, Saccharopolyspora spinosa. Spinosad is a mixture of chemical compounds in the spinosyn family that has a generalized structure consisting of a unique tetracyclic ring system attached to an amino sugar (D-forosamine) and a neutral sugar (tri- Ο -methyl-L-rhamnose). Spinosad is relatively non-polar and not easily dissolved in water.

Mode of action: The mode of action of spinosad is via a neural mechanism. It has both contact and stomach action along with translaminar movement properties. This compound has two unique mode of action, binds primarily with nicotinic acetylcholine receptors and secondarily with GABA receptors which leads to disruption of acetylcholine neurotransmission. Spinosad kills insects via hyperexcitation of the insect nervous system.

[d] Trade name: Award 40 SC

Chemical name: Buprofezin

Empirical formula: C16H23N3OS

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Figure 5. Structural formula of buprofezin

Properties: Buprofezin is a thiadiazine insect growth regulator, especially chitin synthesis inhibitor which inhibits moulting. It is a white crystal and its melting point is 104.5 - 105.5ºC. It is soluble in chloroform, benzene, acetone, ethanol etc. and stable in acidic and alkaline media.

Mode of action: Buprofezin is a thiadiazine like compound that acts as a chitin synthesis inhibitor. It has both contact and vapor phase activity. It inhibits incorporation of 3H-glucosamin into chitin. As a result of chitin deficiency, the procuticle of the insect loses its elasticity and the insect is unable to complete the molting process. Finally, the old cuticle ruptures and insects die. Moreover, it reduces pest populations by reducing fecundity, egg viability, hatchability, increasing egg sterility and making abnormal larvae and pupae by affecting hormonal functions.

Table 1. Specification of the treatments

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3.8 Spray schedule of treatments

Treatments were applied in the brinjal field following the treatments specifications. A total of four sprayings were given at 2 weeks interval. Spraying was done within 9.00 to 11.00 AM to avoid bright sun shine and drift caused by strong wind.

3.9 Data collection

3.9.1 Data collection schedule

In case of shoot and fruit infestation, data were collected on 7 and 14 days after providing each spray. Pre-treatment data were also collected before first spraying. To get marketable and infested fruit yield, fruits were picked at 14 days after treatment application and a total of four pickings were done.

3.9.2 Data collection parameters and procedures

3.9.2.1 Estimation of % shoot infestation

The counting of total and infested shoots was initiated from the very beginning of the brinjal shoot infestation and a pre-treatment data were collected. After treatment application, data were collected at 7 and 14 DAS (days after spraying). The number of total and infested shoots was counted from each plant/plot. Then the number of total and infested shoots was calculated for each plot (4 plants/plot) and the percentage of infested shoots was calculated using the following formula:

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% infested shoot = Po/Pr X 100

Pr = Total number of shoots per plot

Po = Number of infested shoots per plot

Finally, mean percentage of shoot infestation was calculated for each of the treatments from the three replicated plots.

Moreover, percent protection of shoot infestation over control was calculated for each of the treatment as follows:

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Where,

Po=Mean percent of shoot infestation in control plots

Pr =Mean percent of shoot infestation in treated plots

3.9.2.2 Estimation of % fruit infestation

The mean percentage of fruit infestation was calculated after each picking of brinjal fruits. Fruits were collected after 14 days of each spraying and then infested fruits were separated from the total fruits. After that, total and infested fruits were counted carefully per plot and damage was calculated using the following formula:

% infested fruit = Po/Pr × 100

Where,

Po = Total number of infested fruits per plot Pr =Total number of fruits per plot

Finally, mean percentages of fruit infestation for each treatment was calculated from the three replicated plots. Then percent protection of fruit infestation over control for each of the treatment was calculated as follows:

% fruit protection over control = (Po-Pr)/Po × 100

Where,

Po=Mean percent of fruit infestation in control plots

Pr =Mean percent of fruit infestation in treated plots

3.9.2.3 Estimation of the yield (t/ha) of marketable fruits

Marketable brinjal fruits were defined as the visibility of no hole or even no deformation on the fruits. Brinjal fruits were harvested after 14 days of spraying and then marketable fruits were separated carefully from total fruits. After that weight of marketable fruits was taken from the three replicated plots regarding the treatments and yield was converted in ton/hectare. Finally, total marketable yield was taken from all the four pickings. Finally, percent increase in marketable fruit yield over control was calculated for each of the treatment as follows:

% increase in marketable fruit yield over control = (Pr-Po)/Po × 100

Where,

Po = Total amount of marketable fruit yield (t/ha) in control plots

Pr = Total amount of marketable fruit yield (t/ha) in treated plots

3.9.2.4 Estimation of the yield (t/ha) of infested fruits

If any fruit was found with hole or deformation then it was considered as the infested fruit. Infested fruits were separated carefully from healthy fruits. After that weight of infested fruits was taken from the three replicated plots regarding the treatments and yield was converted in ton/hectare. Finally, total infested yield was taken from all the four pickings.

3.10 Statistical analysis of data

The recorded data were compiled and tabulated for statistical analysis. Analysis of variance was done with the help of computer package MSTAT. The mean differences among the treatments were adjudged with Duncan's Multiple Range Test (DMRT) and Least Significant Difference (LSD) when necessary.

3.11 Experimental methods for laboratory study

The efficacy of different doses of buprofezin was evaluated on the mortality, weight reduction and cuticular deformations of brinjal shoot and fruit borer, L. orbonalis (Guen.) larvae in the laboratory, Department of Entomology, Bangladesh Agricultural University. The study was conducted from the period of July to September 2014. Experimental procedures for the laboratory study are described below:

3.11.1 Mass rearing of L. orbonalis

Mass rearing of brinjal shoot and fruit borer was done under laboratory conditions to meet the experimental requirements. Infested brinjal fruits were collected from the field and then larvae were carefully collected by dissecting them. After that larvae were transferred into plastic boxes based on the larval stages and immediately provided with sliced potato tubers (alternate host) as their feed. When larvae were transformed into 5th instars, they came out of tubers by making hole and were usually characterized by their large size and bright-reddish color. After that 5th instars larvae were provided with dry brinjal leaves for pupation. Then the pupae were separated carefully and kept in plastic boxes by covering with a mosquito net to prevent escaping of adults.

Then male and female adults (approximately 10 pairs) were transferred into an artificially made oviposition chamber for mating and egg laying. The inner surface of the oviposition chamber was lined with green paper as well as mosquito net for better egg laying. Before transferring adults into the oviposition chamber, a brinjal seedling was kept inside the chamber for enhancing egg laying by the moths. 5% dextrose was also provided inside the chamber as the feed for moths. After mating, eggs were laid mostly on the ventral surface of the brinjal leaves and some were on green paper and mosquito nets. Egg containing leaves, papers and nets were separated carefully and then kept in petridishes. Eggs were hatched within 2-3 days after oviposition and neonate larvae were immediately placed on sliced potato tubers with fine brush. In this way, 2-3 generations were reared to get enough larvae. 2nd instars larvae were used for all experiments.

3.11.2 Specifications of treatments

Laboratory experiments were consisted of three treatment combinations. Three doses of buprofezin (Award 40 SC, Square Pharmaceuticals Ltd.) i.e. 200, 400 and 800 ppm were provided as treatments. Each treatment was replicated thrice and ten larvae of brinjal shoot and fruit borer were used for each replication. Simultaneously, water treated larvae were placed on fungicide (dithane M-45) soaked potato tubers as a control treatment and replicated for thrice in different application methods.

3.11.3 Treatments application methods

All the treatments were applied through three different methods.

3.11.3.1 Topical application:

In this method, larvae were directly treated (using micropipette) with different concentrations of buprofezin while potato tubers were soaked in 0.2% dithane M-45 to protect from fungal infection and dried on tissues. The treated larvae were then transferred on the dithane M-45 soaked potato slices and finally placed in plastic container having a moist filter paper to avoid desiccation.

3.11.3.2 Potato-dip method:

In this method, sliced potato tubers were first soaked in 0.2% dithane M-45 solution and then dried on tissues. After that potato tubers were treated with different concentrations of buprofezin and further dried on tissues. Then the untreated larvae were transferred on the buprofezin treated potato slices and finally placed in plastic container having a moist filter paper to avoid desiccation.

3.11.3.3 Combination (topical + potato-dip) method:

In this method, buprofezintreated larvae were transferred on buprofezin treated potato slices and placed in plastic container having a moist filter paper to avoid desiccation. Potato slices were previously soaked in 0.2% dithane M-45 to protect the tubers from fungal infection.

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Plate 7. Representative photographs (A-C) showing consecutive steps for topical application method. [A] 2nd instars larvae were treated with different concentrations of buprofezin, [B] Untreated potato slices, [C] Treated larvae were released on the untreated potato slices.

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Plate 8. Representative photographs (A-C) showing the consecutive steps for potato-dip method. [A] Potato slices were dipped in buprofezin solution approximately for 30 seconds, [B] Treated slices were dried on tissues, [C] Untreated larvae were released on treated potato slices.

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Plate 9. Representative photographs (A-D) showing the successive steps for combination method. [A] Potato slices were dipped in buprofezin solution approximately for 30 seconds, [B] Treated slices were dried on tissues, [C] Larvae were treated with buprofezin, [D] Treated larvae were released on treated potato slices.

3.12 Data collection

Data on the mortality was observed at 3 HAT (hours after treatment), 3 and 7 DAT (days after treatment) application. Larval weight was measured at 3 and 7 DAT. Cuticular deformation was observed at 7 DAT. Based on the experimental time-frame, potato slices were dissected very carefully and data were recorded. Died larvae were separated and alive larvae were further provided with fresh/treated potato slices based on the treatment application method.

The percentage of larval mortality and weight reduction was calculated using the following formulae;

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Where,

Po = Number of larvae died

Pr = Number of treated or untreated larvae provided

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Where,

Po = Mean weight of a single larva in control treatment Pr = Mean weight of a single larva in a specific treatment

3.13 Statistical analysis

The recorded data were compiled and tabulated for statistical analysis. Analysis of variance (ANOVA) was done with the help of computer package MSTAT. The mean differences among the treatments were adjudged with Duncan's Multiple Range Test (DMRT) and Least Significant Difference (LSD).

CHAPTER IV RESULTS AND DISCUSSION

A series of field experiments were conducted to evaluate the relative efficacy of microbial and IGR-based biopesticides against the infestation of brinjal shoot and fruit borer (BSFB) (Leucinodes orbonalis Guen.). Treatments were applied individually or in some selected combinations and their effects were evaluated on different parameters viz. percent shoot infestation, percent fruit infestation, infested fruit yield (t/ha), marketable fruit yield (t/ha) etc. Simultaneously, the effect of buprofezin (a chitin synthesis inhibitor) was observed on the mortality, weight reduction and cuticular deformations of BSFB larvae under laboratory conditions. The result of these experiments has been presented and discussed experiment-wise under the following sub headings.

4.1 Effectiveness of selected biopesticides on percent shoot infestation

The percentage of shoot infestation was significantly (P<0.01) reduced when brinjal plants were treated with microbial and IGR-based biopesticides (Table 2). It was observed that each of the treatments was significantly effective against the infestation caused by brinjal shoot and fruit borer. The highest percentage of shoot infestation was observed in case of untreated control which was ranged from 11.49% to 49.04% where the cumulative mean of infestation was 29.06% (Table 2). Shoot infestation caused by L. orbonalis at control plots increased very rapidly with increasing time whereas all other treatments significantly reduced the rate of shoot infestation over time as compared to control.

In this study, three bacterial-fermented biopesticides viz. emamectin benzoate, abamectin and spinosad were evaluated against the shoot infestation caused by BSFB (Table 2). The data clearly showed that all the microbial biopesticides had significant (P<0.01) effect on the reduction of percent shoot infestation compared to the control while differences were insignificant among themselves. The lowest percentage of shoot infestation was observed from emamectin benzoate (8.59%) treated plots which was followed by abamectin (9.01%) and spinosad (10.10%), respectively. Moreover, 70.44, 69.00 and 65.24% shoot was protected from larval infestation when brinjal plants were treated with emamectin benzoate, abamectin and spinosad, respectively (Fig. 6). The results clearly indicated that emamectin benzoate, abamectin and spinosad were effective in reducing percent shoot infestation. Similar results had been found by some other researchers in case of other lepidopteran pests. Udikeri et al. (2004) reported that emamectin benzoate 5 SG at 11 g a.i/ha resulted in significantly lowest larval population (0.10/plant) of cotton bollworm. The damage due to bollworm infestation was the least in emamectin benzoate (4.19%) treated plants.

In this study, the effect of buprofezin (1 and 2 ml/L) was evaluated on the reduction of shoot infestation and both doses were found effective (Table 2). In case of buprofezin (1 ml/L), percentage of shoot infestation was 24.04% and it protected 17.27% shoot from larval infestation as compared to control. When the same biopesticide was applied in a higher dose (2ml/L) the percentage of shoot infestation was 24.04% and it provided 45.18% shoot protection over control and it was also significantly different from the lower dose.

A lower dose of buprofezin (1 ml/L) was applied in combination with emamectin benzoate, abamectin and spinosad and their combined effects on the reduction of shoot infestation was also evaluated (Table 2). It was observed that mean percentage of shoot infestation in buprofezin + emamectin benzoate, buprofezin + abamectin and buprofezin + spinosad treated plots was 8.50, 9.02 and 9.09 percent, respectively. Interestingly, the percentage of shoot infestation was not reduced remarkably when emamectin benzoate, abamectin and spinosad were applied in combination with buprofezin (1 ml/L) (Table 2). More clearly, 70.44, 69.00 and 65.24% shoot was protected from larval infestation when emamectin benzoate, abamectin and spinosad were sprayed individually whereas 70.75, 68.96 and 68.72% shoot protection was achieved from buprofezin + emamectin benzoate, buprofezin + abamectin and buprofezin + spinosad treated plots, respectively (Fig. 6). The results indicated that the combined action was either slightly additive or antagonistic.

Therefore, the results of percent shoot infestation clearly revealed that all three microbial biopesticides were moderately effective against BSFB in reducing shoot infestation and infestation was reduced in slightly greater percentage when these biopesticides were applied in combination with buprofezin (1 ml/L). Buprofezin 1ml/L was found less effective while higher concentration (2 ml/L) was slightly more effective to reduce shoot infestation and it was found significant over the lower dose 1ml/L.

These findings could be linked with Kumar and Devappa (2006) who reported that when proclaim 5 SG (emamectin benzoate) was tested against brinjal shoot and fruit borer during 2002-2003 and 2003-2004, it was found most effective among the treatments. The results of their experiment indicated that the application of proclaim 5 SG @ 200 g/ha was found most effective in reducing the dead hearts and also fruit damage in brinjal. The total yield was also higher in this treatment.

Table 2. Mean percentage of shoot infestation caused by L. orbonalis at different sprayings against different treatments

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In a column, means followed by similar letter(s) are not significantly different. [DAS: Days After Spraying]

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Figure 6. Percent protection of brinjal shoots over control when brinjal plants were treated with selected microbial and IGR-based biopesticides either singly or in some selected combinations. Maximum protection of shoot infestation was observed in case of buprofezin + emamectin benzoate treated plots which were followed by other treatments. The lowest reduction was obtained from buprofezin 1ml/L treated plots.

[Here, Bupro means Buprofezin ; EB means Emamectin benzoate]

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Plate 10. A healthy brinjal plant with fruits

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Plate 11. An infested brinjal shoot

4.2 Effectiveness of selected biopesticides on percent fruit infestation

The effects of microbial and IGR-based biopesticides on percent fruit infestation has shown in Table 3. Similar to shoot protection, all the selected biopesticides significantly (P<0.01) reduced percent fruit infestation in comparison with control treatment.

The highest percentage of infested fruits from all 4 pickings was obtained when brinjal plants were left untreated (19.46, 27.85, 30.62 and 28.57% after first, second, third and fourth picking, respectively) where the cumulative mean was found 26.63 percent (Table 3). This comparatively lower fruit infestation raised the possibility that the selected variety (Amjhuri) was moderately resistant to BSFB infestation because literature reveled that about 50 to 70% fruit infestation was recorded from some other brinjal varieties like Jhumki. Ali and Rahman (1980) made a brief observation on the incidence of shoot and fruit borer on 12 cultivars of brinjal. He observed that the lowest percentage of fruit infestation (25%) occurred in Singnath and the highest (86%) in Jhumki.

The percentage of fruit infestation was significantly reduced when brinjal plants were treated with selected biopesticides viz. emamectin benzoate, abamectin, spinosad and buprofezin either alone or in some chosen combinations (Table 3). The results indicated that all three microbial biopesticides viz. emamectin benzoate, abamectin and spinosad showed better efficacy than IGR-based biopesticide buprofezin regarding the percentage of fruit infestation. More clearly, 11.03% mean fruit infestation was recorded when brinjal plants were treated with emamectin benzoate which was followed by abamectin (12.10%) and spinosad (12.51%), respectively. Therefore, 58.58, 54.26 and 53.02% fruits were protected from larval infestation when brinjal plants were treated with emamectin benzoate, abamectin and spinosad, respectively (Fig. 7).

The effect of buprofezin on the reduction of percent fruit infestation was found statistically significant (P<0.01) (Table 3). The mean percentage of fruit infestation in buprofezin 1ml/L treated plots was recorded 22.46% which were followed by buprofezin 2 ml/L treated plots (16.35%) and there had significant difference between two doses of buprofezin. Although the highest percentage of infested fruits after first picking was obtained from buprofezin 1ml/L treated plots, this treatment provided some sort of protection from second spraying and the percentage of fruit protection ultimately became 15.66% as compared to control treated plots. When the same biopesticide was applied in higher dose (2 ml/L) as another treatment it provided 38.66% fruit protection over control and it was the second worst treatment after buprofezin (1 ml/L) (Fig. 7).

These findings could be linked with the observations of Nasr et al. (2010) who found that buprofezin caused reasonable mortality in Spodoptera littoralis larvae. Ragaei and Sabri (2011) also found that buprofezin was moderately effective against the fourth instars larvae of the cotton leafworm, Spodoptera littoralis and caused significant mortality and growth reduction of the leafworm larvae.

The combination effect of emamectin benzoate, abamectin and spinosad when applied in combination with 1 ml/L buprofezin was also evaluated on percent fruit infestation. Interestingly, percent fruit infestation was not reduced remarkably when these microbial biopesticides were applied in combination with buprofezin (Table 3). It was observed that mean percentage of fruit infestation in buprofezin + emamectin benzoate, buprofezin + abamectin and buprofezin + spinosad treated plots was 9.59, 10.80 and 12.19 percent, respectively. More clearly, 63.99, 59.44 and 54.22% fruit protection was obtained from buprofezin + emamectin benzoate, buprofezin + abamectin and buprofezin + spinosad treated plots, respectively. It is mentionable that the treatment buprofezin + emamectin benzoate provided more significant effect compared to other two combinations. The reason was not clear but it raised the possibility that there was better synergistic action between emamectin benzoate and buprofezin than others.

These findings could be linked with the experimental results of Udikeri et al. (2004), who reported that emamectin benzoate 5 SG at 11 g a.i/ha resulted in significantly the lowest larval population (0.10/plant) of cotton bollworm and was found at par with spinosad 48 SC @ 50 g a.i/ha (0.14/plant). The damage due to bollworm was least in emamectin benzoate treated plants (4.19%), which resulted in significantly more number of good opened bolls and less number of bad opened bolls along with the highest seed cotton yield (15.93 q/ha). Bhemanna et al. (2005) also evaluated emamectin benzoate (proclaim 5% SG), a new insecticide against okra fruit borers. Emamectin benzoate @ 8.50 g a.i/ha recorded lower fruit borer damage and higher fruit yield and was highly promising against okra fruit borer complex.

Table 3. Mean percentage of fruit infestation by L. orbonalis at different pickings against different treatments

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In a column, means followed by similar letter(s) are not significantly different.

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Figure 7. Percent fruit protection over control when brinjal plants were treated with selected microbial and IGR based biopesticides either singly or in some selected combinations. Maximum fruit protection was observed from buprofezin + emamectin benzoate treated plots which was followed by other treatments. The lowest fruit protection was obtained from buprofezin 1ml/L treated plots.

[Here, Bupro means Buprofezin ; EB means Emamectin benzoate]

4.3 Effects of selected biopesticides on the yield of marketable fruits (t/ha)

In this study, the efficacy of selected biopesticides on marketable fruit yield (t/ha) was evaluated and their yield performances were shown in Table 4. Among three bacterialfermented biopesticides, the maximum marketable yield was obtained from emamectin benzoate treated plots (9.86 t/ha) which was followed by abamectin (9.34 t/ha) and spinosad (8.17 t/ha), respectively (Table 4). It was also estimated that about 83.96, 74.25 and 52.43% marketable fruit yield was increased over control when brinjal plants were treated with emamectin benzoate, abamectin and spinosad, respectively (Fig. 8). Moreover, about 31% and 9% fruit yield was increased in emamectin benzoate treated plots compared to spinosad and abamectin, respectively.

Both doses of buprofezin significantly increased marketable fruit yield as compared to control (Table 4). It was observed that total marketable fruit yield was 7.36 t/ha in case of buprofezin 2 ml/L treated plots while 6.05 t/ha in case of buprofezin 1 ml/L treated plots. Although the marketable fruit yield in buprofezin treated plots was statistically significant compared to control but both the doses (1 and 2 ml/L) were worst treatments as compared to other biopesticides. Only 12.87% marketable fruit yield was increased over control when brinjal plants were treated with 1 ml/L buprofezin whereas the yield was increased about 3 times (37.31%) when buprofezin was applied in concentration of 2 ml/L and this higher dose was significantly different from the lower dose.

The marketable fruit yield was increased very slightly compared to individual treatments when emamectin benzoate, abamectin and spinosad were applied in combination with buprofezin (1 ml/L). However, the highest amount of marketable fruit yield was obtained from buprofezin + emamectin benzoate treated plots (9.94 t/ha) which was followed by buprofezin + abamectin (9.88 t/ha) and buprofezin + spinosad (8.81 t/ha) treated plots, respectively. The application of buprofezin + emamectin benzoate, buprofezin + abamectin and buprofezin + spinosad increased 85.45, 84.33 and 64.37% marketable fruit yield over control, respectively (Fig. 8).

It was also noted that the treatment spinosad provided comparatively poorer efficacy than emamectin benzoate and abamectin when spinosad was applied individually or in combination with buprofezin (Table 4 and Fig. 8). There was insignificant differences between emamectin benzoate and abamectin regarding fruit yield (individual or combined effect) while these treatments significantly differed from spinosad. Regarding marketable fruit yield, emamectin benzoate and abamectin provided the highest yield when they were applied individually or in combination with buprofezin and this result was followed by buprofezin + spinosad, spinosad, buprofezin 2 and 1 ml/L, respectively. The lowest yield was recorded from untreated plots.

These findings could be linked with some previous research works. Anil and Sharma (2010), studied on the efficacy of spinosad and emamectin benzoate against BSFB. They found that spinosad and emamectin benzoate were effective in suppressing the fruit infestation by BSFB. Murugaraj et al. (2006) reported that emamectin benzoate (proclaim 5 SG) @ 11 g a.i/ha was highly effective in reducing the larval population of H. armigera, fruit damage as well as in increasing the tomato yield.

Ashok et al. (2001) studied the effect of some novel insecticides against P. xylostella. Results showed that novaluron 0.00075 percent was found to be most effective in increasing marketable fruit yield followed by abamectin 0.00045 percent and diafenthiuron 0.12 percent.

Table 4. Yield of marketable brinjal fruits after application of different treatments

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In a column, means followed by similar letter(s) are not significantly different.

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Figure 8. Increase in percent marketable fruit yield over control when brinjal plants were treated with selected microbial and IGR-based biopesticides either singly or in some selected combinations. Maximum increase in marketable fruit yield over control was obtained from buprofezin + emamectin benzoate treated plots which was followed by other treatments. The lowest increase in percent marketable fruit yield was obtained from buprofezin (1 ml/L) treated plots.

[Here, Bupro means Buprofezin ; EB means Emamectin benzoate]

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Plate 12. Some infested brinjal fruits after picking

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Plate 13. An infested brinjal fruit with larvae inside

4.4 Effects of selected biopesticides on the yield of infested fruits (t/ha)

It was observed that each of the treatments was significantly effective against brinjal shoot and fruit borer infestation and reduced infested fruit yield as compared to control (Table 5). Among three bacterial-fermented biopesticides, the lowest amount of infested fruit yield was obtained from emamectin benzoate treated plots (1.12 t/ha) which was followed by abamectin (1.19 t/ha) and spinosad (2.03 t/ha), respectively. However, treatment emamectin benzoate and abamectin were statistically insignificant between them. The infested fruit yield was reduced slightly as compared to the individual treatment when emamectin benzoate and abamectin were applied in combination with buprofezin 1ml/L (0.79 and 0.80 t/ha, respectively). On the other hand, spinosad provided comparatively poorer efficacy than emamectin benzoate and abamectin when it was applied in combination with buprofezin (1.41 t/ha infested fruit yield).

Moderate amount of infested fruit yield was obtained from the plots that were treated with 2 ml/L of buprofezin (2.76 t/ha) and it was followed by 1 ml/L of buprofezin (3.06 t/ha) and these two doses differed significantly. The highest amount of infested fruit yield was recorded from untreated control plots (3.47 t/ha).

It was observed that there was synchronization between the reduction in infested fruit yield and increase in marketable fruit yield for each of the treatment. The treatment which caused maximum reduction in infested fruit yield as compared to control resulted in highest amount of marketable fruit yield as in case of buprofezin + emamectin benzoate and buprofezin + abamectin treated plots. Similarly, those treatments caused minimum reduction in infested fruit yield over control resulted in lesser amount of marketable fruit yield as in buprofezin @1ml/L and 2 ml/L treated plots.

Table 5. Yield of infested brinjal fruits after application of different treatments

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In a column, means followed by similar letter(s) are not significantly different.

4.5 Comparative efficacy of different doses of buprofezin on the mortality, weight reduction and cuticular deformation of brinjal shoot and fruit borer larvae

Experiments were conducted in the laboratory to evaluate the efficacy of buprofezin on the mortality as well as weight reduction of L. orbonalis larvae . Cuticular deformation of larva was also observed following application of treatments. Different doses of Buprofezin (Award 40 SC) viz. 200, 400 and 800 ppm were applied through different application methods like topical (direct), potato-dip (indirect) and combination (topical + potato-dip) method. It was actually done to understand whether buprofezin has any systemic action or not and whether it is appropriate for internal feeder. The result of these experiments has been presented and discussed experiment-wise under the following sub headings.

4.5.1 Efficacy of different doses of buprofezin on the mortality and weight reduction of L. orbonalis through topical application method

4.5.1.1 Effects on larval mortality

The topical application of different doses of buprofezin on the mortality of brinjal shoot and fruit borer larvae has shown in the Table 6 (P<0.01). The results clearly revealed that buprofezin was moderately effective against L. orbonalis and the effect was clearly dose-dependent. No mortality was found at 3 hours after treatment application, the significant effect was found at 3 DAT (P<0.01) which was consistent up to 7 DAT (P<0. 01). The maximum, 44.44% mortality was recorded from 800 ppm which was followed by 400 (28.28%) and 200 ppm (16.30%) of buprofezin, respectively. The lowest mortality (16.30%) was found from 200 ppm which was almost comparable with that of water- treated control (11.11%) and both of them were statistically insignificant.

Table 6. Mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

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In a column, means followed by similar letter(s) are not significantly different. [NS: Not significant; HAT: Hours after treatment; DAT: Days after treatment]

4.5.1.2 Effects on larval weight

The weight of larvae gradually decreased when larvae were directly treated with buprofezin (Table 7). Among the different concentrations of buprofezin the lowest mean larval weight was obtained from 800 ppm (35.87 mg/larva) which was followed by 400 pm (41.43 mg/larva) and both these doses had significant effect on larval weight. However, mean larval weight in case of the lowest dose of buprofezin (200 ppm) was statistically insignificant with the water treated control larvae (54.07 vs. 56.98 mg/larva, respectively). More clearly, the maximum weight reduction of larvae (37.04%) was observed from 800 ppm of buprofezin which was followed by 400 ppm (27.29%) and 200 ppm (5.10%), respectively as compared to the water treated control (Fig. 9).

Table 7. Weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

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In a column, means followed by similar letter(s) are not significantly different. [NS: Not significant; HAT: Hours after treatment; DAT: Days after treatment]

4.5.2 Efficacy of different doses of buprofezin on the mortality and weight reduction of L. orbonalis through potato-dip method

4.5.2.1 Effects on larval mortality

Significant larval mortality was found when untreated larvae were provided with buprofezin treated potato slices (P<0.01) (Table 8). It was observed that the potato-dip method was comparatively more effective than topical application method regarding mortality percentages. The trend of buprofezin effect was similar with that of topical application method. Specifically, the maximum 66.66% mortality was recorded from 800 ppm which was followed by 400 ppm (43.33%) and 200 ppm (33.33%) of buprofezin, respectively. It was interesting observation that the lowest concentration had significant effect on the larval mortality (33.33%) in case of potato-dip method while topical application method had insignificant effect as compared to water treated control. The

lowest mortality was recorded from untreated control (13.33%) where the untreated larvae were simply placed in untreated potato slices.

Table 8. Mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through potato-dip method

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In a column, means followed by similar letter(s) are not significantly different. [NS: Not significant; HAT: Hours after treatment; DAT: Days after treatment]

4.5.2.2 Effects on larval weight

Buprofezin had dose dependent effects on the reduction of larval weight (Table 9). The weight of larvae was gradually decreased with increasing concentration level and time duration. Among the different concentrations of buprofezin the lowest mean larval weight was obtained from 800 ppm (12.37 mg/larva) which was followed by 400 pm (27.03 mg/larva) and 200 ppm (43.57 mg/larva), respectively. Here, mean larval weight in case of all the doses of buprofezin was signficantly different as compared to water treated control larvae. The maximum mean weight of larva (61.75 mg/larva) was found in water treated control. Interestingly, the lowest concentration of buprofezin (200 ppm) had significant effect on the larval weight in this potato-dip application method while topical application method had insignificant effect. The maximum reduction in larval weight was observed on 7 DAT. Approximately 80% weight reduction was found when larvae fed treated potato with 800 ppm concentration of buprofezin followed by 400 ppm (56.23%) and 200 ppm (29.44%), respectively compared to water treated control larvae (Fig. 9).

Table 9. Weight of L. orbonalis larvae at different time interval after treating with with different concentrations of buprofezin through potato-dip method

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In a column, means followed by similar letter(s) are not significantly different. [NS: Not significant; HAT: Hours after treatment; DAT: Days after treatment]

4.5.3 Comparative efficacy of different doses of buprofezin on the mortality and weight and reduction of L. orbonalis through combination (topical + potato-dip) method

4.5.3.1 Effects on larval mortality

The highest larval mortality was found when larvae and potato slices both were treated with buprofezin compared to individual treatment (Table 10). No mortality was found at 3 hours after treatment application but significant mortality of the larvae was found at 3 DAT which further increased at 7 DAT. At 7 DAT, the maximum mortality was found from 800 ppm (69.44%) of buprofezin which was followed by 400 ppm (50.0%) and 200 ppm (36.67%), respectively (Fig. 9). The significant mortality was found even from 200 ppm (36.67%) of buprofezin in comparison with that in the water-treated control. The lowest mortality was recorded from water treated control (12.7%) where the larvae were placed on untreated potato slices.

Table 10. Mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

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In a column, means followed by similar letter(s) are not significantly different. [NS: Not significant; HAT: Hours after treatment; DAT: Days after treatment]

4.5.3.2 Effects on larval weight

Buprofezin had significant and dose dependent effects on larval weight (P<0.01). Among the different concentrations of buprofezin the lowest mean larval weight was obtained from 800 ppm (10.88 mg/larva) which was followed by 400 pm (24.03 mg/larva) and 200 ppm (41.13 mg/larva), respectively. Here, mean weight of larvae in case of all the doses of buprofezin was signficantly different as compared to water treated control larvae. The maximum mean weight of larvae (63.19 mg/larva) was found in water treated control. The pattern of larval weight reduction was similar with that of potato-dip method although reduction level was higher than potato-dip method (Table 11). It was observed that larval weight reduced gradually with increasing time and the effect was clearly dose dependent. Approximately 83% weight reduction was observed from 800 ppm of buprofezin which was followed by 400 ppm (61.97%) (Fig. 9). About 35% weight was reduced when both larvae and potato tubers were treated with lower concentrations (200 ppm) of buprofezin as compared to the water-treated control.

Table 11. Weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

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In a column, means followed by similar letter(s) are not significantly different. [NS: Not significant; HAT: Hours after treatment; DAT: Days after treatment]

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Figure 9. Percent weight reduction of L. orbonalis larvae over control. Second instars larvae were treated with different concentrations of buprofezin through different application methods and weight reduction was observed at 3 and 7 DAT. Weight reduction was clearly dose and method dependent. Maximum reduction in larval weight was observed when both larvae and potato slices (combined method) were treated with 800 ppm of buprofezin which was followed by potato-dip method. Topical method as well as lower dose of buprofezin (200 ppm) was less effective.

4.6 Effects on cuticular deformations

Buprofezin is a chitin synthesis inhibitor i.e. it affects moulting process by inhibiting chitin bio-synthesis. In this study it was observed that buprofezin potently inhibits chitin synthesis and thereby the colour of cuticle was changed and cuticle was fractured (Plate 15). Second instars larvae were treated with different concentrations of buprofezin following different application methods whereas control larvae were treated with water. Cuticular deformations and color changes were observed at 7 DAT (days after treatment application).

Maximum cuticular deformation was observed when larvae were treated with higher concentrations of buprofezin through potato-dip and combination methods. A comparatively weak change was found among the larvae that were treated with 200 ppm of buprofezin as compared to 400 and 800 ppm. However, no cuticular deformation was found when water treated larvae were placed in untreated potato tubers which clearly indicated that cuticular deformation was entirely caused by the actions of buprofezin.

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Plate 14. Experiment in the laboratory

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Plate 15. Representative photomicrographs of the larvae of Leucinodes orbonalis after 7 days of treatment (DAT) application. [A] 2nd instars larvae that were treated with water, [B] larvae that were treated with 800 ppm of buprofezin, [C] An untreated larva with normal cuticle, [D] A treated larva with cuticular deformations. Severe cuticular deformation and colour changes were observed when larvae were treated with 800 ppm of buprofezin while no changes were observed when treated with only water.

CHAPTER V SUMMARY AND CONCLUSION

A series of experiments were conducted in the Entomology Field Laboratory, Bangladesh Agricultural University (BAU), Mymensingh during January-July, 2014 to evaluate the relative efficacy of microbial and IGR-based biopesticides against the infestation of brinjal shoot and fruit borer, Leucinodes orbonalis Guen. Concurrently, effects of buprofezin (Insect Growth Regulator) on the mortality, weight reduction and cuticular deformations of BSFB larvae were observed under laboratory conditions. The summarized results and conclusion of the study are as follows:

The present study showed that each of the biopesticidal treatments was significantly effective against the shoot infestation caused by brinjal shoot and fruit borer. The highest percentage of shoot infestation was found in control treated plots (29.06%). Among the treatments the lowest percentage of shoot infesatation was found in buprofezin + emamectin benzoate treated plots (8.50%) which was followed by emamectin benzoate (8.59%), abamectin (9.01%), buprofezin + abamectin (9.02%), buprofezin + spinosad (9.09%), spinosad (10.10%), buprofezin 2 ml/L (15.93%) and buprofezin 1ml/L (24.04%), respectively.

From the experimental data it was observed that the highest percentage of fruit infestation in case of all four pickings was obtained when brinjal plants were left untreated and mean fruit infestation was found 26.63%. However, all other treatments were found significantly effective in reducing percent fruit infestation over control. The lowest percentage of fruit infestation was found in buprofezin + emamectin benzoate (9.58%) treated plots which was the best treatment in reducing percent fruit infestation and it was followed by buprofezin + abamectin (10.80%), emamectin benzoate (11.03%), abamectin (12.10%), buprofezin + spinosad (12.19%), spinosad (12.51%), buprofezin 2 ml/L (16.35%) and buprofezin 1ml/L (22.46%), respectively.

In this study, the efficacy of selected bio-pesticides on marketable fruit yield (t/ha) was evaluated and it was found that all the treatments significantly increased marketable fruit yields as compared to control. The maximum amount of marketable fruit yield was obtained from buprofezin + emamectin benzoate treated plots (9.94 t/ha) which was followed by buprofezin + abamectin (9.88 t/ha), emamectin benzoate (9.86 t/ha), abamectin (9.34 t/ha), buprofezin + spinosad (8.81 t/ha), spinosad (8.17 t/ha), buprofezin 2 ml/L (7.36 t/ha) and buprofezin 1 ml/L (6.05 t/ha) treated plots, respectively. However, lowest amount of marketable yield was obtained from the untreated brinjal plots (5.36 t/ha).

The experimental results indicated that each of the treatments were significantly effective in reducing infested fruit yield as compared to control. The lowest amount of infested fruit yield was obtained from buprofezin + emamectin benzoate treated plots (0.79 t/ha) which was followed by buprofezin + abamectin (0.80 t/ha), emamectin benzoate (1.12 t/ha), abamectin (1.19 t/ha), buprofezin + spinosad (1.41 t/ha), spinosad (2.03 t/ha), buprofezin 2 ml/L (2.76 t/ha) and buprofezin 1 ml/L (3.06 t/ha) treated plots, respectively. However, highest amount of infested fruit yield (3.47 t/ha) was obtained when brinjal plants were left untreated.

The laboratory experiments clearly revealed that for different application methods all the three doses of buprofezin i.e. 200, 400 and 800 ppm were significantly effective against L. orbonalis larvae in terms of both mortality and weight reduction. No mortality was found immediately after buprofezin application (3HAT) in case of all the three doses irrespective of application methods. However, the mortality was significantly increased at 3 days after treatment (DAT) application that further increased at 7 DAT. It was observed that the mortality was clearly dose-dependent and the highest percentage of larval mortality was found at 7 DAT when buprofezin concentration was 800 ppm (44.44%, 66.66% and 69.44% mortality in case of topical, potato-dip and topical+ potato- dip methods, respectively) followed by 400 ppm (28.28%, 43.33% and 50.00% mortality in case of topical, potato-dip and topical+ potato-dip methods, respectively) and 200 ppm (16.30%, 33.33% and 36.67% mortality in case of topical, potato-dip and topical+ potato-dip methods, respectively). The lowest percentage of larval mortality was found in water treated control (11.11%, 13.33% and 12.70% mortality in case of topical, potatodip and topical+ potato-dip methods, respectively).

Like as mortality, the growth of larvae was significantly inhibited by buprofezin and that was also dose-dependent. The highest percentage of larval weight reduction over control was found at 7 DAT when larvae were treated with buprofezin in 800 ppm concentration through different treatment application methods (37.04%, 79.96% and 82.78% weight reduction in case of topical, potato-dip and topical+ potato-dip methods, respectively) followed by 400 ppm (27.29%, 56.23% and 61.97% weight reduction in case of topical, potato-dip and topical+ potato-dip methods, respectively) and 200 ppm (5.10%, 29.44% and 34.91% weight reduction in case of topical, potato-dip and topical + potato-dip methods, respectively). The cuticular deformation was clearly observed when larvae were treated with buprofezin following different application methods which also suggested that the target of buprofezin was cuticle and buprofezin had hampered cuticule formation by blocking endocrine-pathway.

From the experimental findings it can be concluded that all three bacterial-fermented biopesticides viz. emamectin benzoate, abamectin and spinosad can be applied successfully for the management of BSFB as they provided significant protection of brinjal shoots and fruits over control. Although the IGR-based biopesticide, buprofezin provided significant protection of brinjal shoots and fruits as compared to control it should not be used solely for the management of BSFB as the percentage of protection was not so satisfactory. Therefore, buprofezin should be used as a component of an IPM program. However, the combined doses of buprofezin (1 ml/L) with three bacterial fermented biopesticides were slightly more effective than the single doses of each biopesticide but this might cost more as compared to single doses. Moreover, from the laboratory study it was clear that larval mortality and growth reduction was higher when larvae fed buprofezin treated potato than larvae directly treated with buprofezin. It raises the possibility that buprofezin works more effectively when it reaches to the stomach through food consumption than cuticular contact. This finding suggests that moulting or chitin bio-synthesis is an inter-physiological process than simply cuticular process. The findings of stomach action of buprofezin may be helpful to control L. orbonalis in the field conditions as they are internal feeders.

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APPENDIX I

Analysis of variance for mean percentage of shoot infestation caused by L. orbonalis at different sprayings against different treatments

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APPENDIX II

Analysis of variance for mean percentage of fruit infestation by L. orbonalis at different pickings against different treatments

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APPENDIX III

Analysis of variance for the yield of marketable brinjal fruits after application of different treatments

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APPENDIX IV

Analysis of variance for the yield of infested brinjal fruits after application of different

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APPENDIX V

Analysis of variance for mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

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APPENDIX VI

Analysis of variance for mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through potato-dip method

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APPENDIX VII

Analysis of variance for mean percent mortality of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

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APPENDIX VIII

Analysis of variance for mean weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through topical application method

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APPENDIX IX

Analysis of variance for mean weight of L. orbonalis larvae at different time interval after treating with with different concentrations of buprofezin through potato-dip method

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APPENDIX X

Analysis of variance for mean weight of L. orbonalis larvae at different time interval after treating with different concentrations of buprofezin through combined method

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APPENDIX XI

Pattern of mean monthly rainfall, humidity, and temperature during the field experimental period (January to June, 2014)

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Source: Weather Yard, Department of Irrigation and Water Management, BAU Campus Station, Mymensingh.