VDF- Very Dense Forest (>70%), MDF- Moderately Dense Forest (40-70%), OF- Open Forest (10-40%).

Transcription

1 CHAPTER-3 MATERIALS AND METHODS 3.1 Site Selection: A field survey of various localities of Dehradun was carried out to study the forest vegetation and their existence in the field. During the survey, areas of different host plants of targeted pest existence were identified for collection of various stages of the pest and experimental sites where the host plants and pest were present profusely, were marked. After survey, six sites around Dehradun were selected namely Barkot (Roadside plantation of age years), Forest Research Institute Dehradun (Saplings of 3-4 years), Langha (Natural forest), Kalsi (Forest nursery with 1 year seedlings), Timli (Natural forest), and Thano (Natural forest) for general collection of target pest for laboratory experiments (Plate- 1). From these, three sites were marked for seasonal abundance. The sites are located at various distances in and around Dehradun at various longitude and latitude described as below (Map- 1 and 3). (Source- Forest Survey of India) S. No. Site Longitude Latitude Forest type 1. F.R.I E N MDF 2. Barkot E N VDF 3. Langha E N MDF 4. Kalsi E N OF 5. Timli E N VDF 6. Thano E N VDF VDF- Very Dense Forest (>70%), MDF- Moderately Dense Forest (40-70%), OF- Open Forest (10-40%). Other information about these sites like soil, climatic condition, general land use and cropping pattern, irrigation and hydrology and relief and slope, are also shown in map- 3. (Source- Survey of India, Dehradun) 3.2 Survey and Collection: Selected and marked sites were surveyed at periodic intervals (monthly) from September 2006 November 2009 for collection and record of damage due to defoliation by H. talaca in forest nurseries, plantations and natural stands in and around Dehradun.

2 Forest Cover Map Of Dehradun UTTARKASHI SIRMAUR Kalsi Langha TEHRI GARHWAL Timli FRI SAHARANPUR Legends Very Dense Forest(>70%) Moderately Dese Forest ( %) Open Forest( %) Non Forest Scrub Water Thano Barkot HARIDWAR The pest situation and studies were conducted at Kalsi, Thano, Langha, Timli, Barkot, New forest (F.R.I) and multipurpose trees growing in forest, road sides, and parks.

3 (C) (D) (E) Map. 3: (A) Soils. (B) Climatic condition. (C) General landuse and cropping pattern. (D) Irrigation and hydrogeology. (E) Relief and slope. Source: Survey of India Dehradun.

4 (A) (B) (C) (D) Plate- 1 (A) Road side plantation of Cassia glauca. (B) Toon nusrery. (C) Shisham plantation. (D) Out door cage.

5 (C) (D) (E) (F) Plate- 2 (A) Collection of larvae from nursery. (B) Collection of larvae from natural forest. (C) Mass collection of larvae from forest. (D) Collection of larvae in plastic boxes. (E) Collection of pupae from forest soil. (F) Collection of pupae in glass jar.

6 Various stages of defoliator were collected from March to November on various multipurpose trees in the above localities for mass rearing and maintenance of the stock culture. Collection was made by handpicking and sweeping methods in early morning hours. The larvae were collected in plastic boxes & plastic tubes (size 10x11cm) and the open end of the boxes and tubes were covered with markin/ muslin cloth tied with rubber band. The larvae were released inside glass chimney cages, wooden glass cages and outdoor cages with fresh leaves regularly supplied for laboratory studies. Collection of the pupae of H. talaca from the field was done by digging out the soil at the depth of about 2 to 6 cm around different host plants. The soil was dug out carefully to avoid damage because some pupae were observed in the top soil or upper layers of soil. The pupation by last instar larvae is preferred in the soft soil or humus. The mature larvae descend down from the tree crown with the help of silken thread and borrow in the humus in the ground for pupation and complete its pupal development in the soil (Plate- 2). 3.3 Rearing of H. talaca: The larvae were reared in the glass chimney cages, wooden wire mesh cages (size- 60 cmx60 cmx90 cm, sliding glass on one side and the bottom made of wooden plank), as well as in outdoor cages of insectory. For maintaining the laboratory culture, seedlings of different host plants were obtained regularly from Central Nursery, F.R.I. Dehradun. Beside seedlings, fresh leaves were also collected from botanical garden, insectory, road side plantations and central nursery, F.R.I. Seedlings and were kept inside the wire mesh cages. Eggs laid in the beaker, on the leaves or muslin cloth tied at the mouth of the beaker and on the wall of the beaker were kept inside the cage. A branch was bent so that it remains inside the beaker. The newly hatched larvae immediately got their preferred food in the form of seedling with fresh and tender leaves. The older instars (4 th and 5 th ) were shifted to another cages and fresh leaves were provided daily and were placed in water filled round bottom conical flask with their bases dipped in water. The foliage was held in position with the help of cotton which was wrapped around the branches at the mouth of flask. These arrangements were done to prevent early drying of

7 (C) (D) (E) (F) Plate- 3 (A) Rearing of larvae in outdoor cages of insectory on Toona ciliate. (B) Mass rearing of larvae in wooden wire mesh cage on Cassia glauca. (C) Mass rearing of larvae in wooden wire mesh cage. (D) Rearing of larvae on different hosts plants. (E) Rearing of larvae and pupae at room temperature. (F) Rearing of larvae and pupae under controlled conditions.

8 host leaves. Old branches were replaced daily with fresh ones. The rearing was carried out at room temperature (26ºC ± 1ºC) and 80 ± 5 % humidity (Plate- 3). 3.4 Biology of H. talaca: For the study of fecundity, mating behavior, oviposition period, incubation period, larval stages pupal stages and longevity of the adults separate experiments were conducted. In each experiment, ten pairs of moths of H. talaca (male and female) were kept in chimney cages with fresh leaves of various host plants and outdoor cages. Observation on larval development like number of instars, length, colour, molting and feeding behavior were recorded daily. Effect of temperature, humidity on the development of larval stages was also studied. 3.5 Studies of Various Developmental stages: The biological observation on egg, larval period, pupal period, copulation, oviposition, and longevity was recorded periodically for all generations Egg: Each potted plant having eggs of H. talalca was examined thoroughly for number of eggs with the help of hand lens. The eggs were observed regularly for hatching without removing them from the plant. The observations were also recorded on shape, size, colour, texture, pattern of egg deposition, site of egg laying, fecundity, other visible changes in egg, length and width of egg was measured under stereoscopic binocular microscope for thirty eggs. The data on incubation period was recorded on eggs and per cent hatchability was worked out. Mode of hatching was also observed under stereoscopic binocular microscope. Effect of temperature and humidity on the viability and incubation period of the eggs was carried out by keeping freshly laid counted number of eggs in Petri dishes (7.5 cm) lined at the bottom with a filter paper. Petri dishes were stored in desiccators maintaining humidity, which were kept in B.O.D. incubators set at desired temperature. The observations on the viability of eggs and incubation period were recorded.

9 3.5.2 Larva: After hatching of eggs, the newly hatched larvae were transferred inside the small glass tubes (2cmx10cm) covered with the muslin cloth. The fresh and tender leaves were supplied daily as food. The observation on the larvae and their development, duration of each larval stage, the number of instars, size, head width, colour, molting and feeding habit, were recorded. To investigate the larval duration and the factors responsible for larval mortality at different temperature and humidity in the laboratory conditions, counted number of larvae was kept in 500 ml capacity clean glass beakers lined at the bottom with the filter paper. Each such beaker was then covered by muslin cloth tied with the help of rubber band and transferred to desiccators maintained at desired humidity, which were kept in B.O.D. incubators set at desired temperature. In each experiment, three sets were kept. Fresh foliage of host plant was provided daily as food to the larvae. The date of pupation and the number of larvae pupated were noted and larval mortality was also recorded Pupa: Full-fed larvae stopped feeding and took some time for pupal formation. The time interval between cessation of feeding and actual transformation into pupae was considered as pre-pupal period. Twenty full-fed larvae, reared under laboratory conditions, were transferred to glass jars for pupation. The Pupation behavior was studied under three different conditions. Glass jar containing 15 cm deep forest soil and twigs of host plants, glass jar containing 15 cm deep humus and twigs host plants, glass jar containing only tender twigs of host plants. For each treatment, 20 larvae were released and each treatment was replicated thrice. The number of larvae, which changed into pupae was counted daily in each set. Duration of pupal period was recorded replication wise in each treatment separately. The pupae were sexed and observation on color, length x breadth (n = 20) was recorded sex-wise. Observations on the position of genital openings and anal opening in male and female were recorded under stereoscopic binocular microscope for sexing.

10 The effect of temperature and humidity on pupal period was worked out by keeping these beakers in desiccators maintaining different humidity, which were then kept at different temperatures in BOD. Observations on the emergence of moths were recorded to calculate pupal duration. Adult emergence from field collected pupae was recorded at an interval of 4 hours. To study the mode of moth emergence, the pupae were kept in glass jars with its bottom lined with moist foam sheet (9.0 x 2.5 cm) Mating behavior, copulation and egg laying: Freshly emerged moths were collected in glass tubes. Male and female moths emerging on the same day were paired and released into the cages. A Cotton swab dipped in ten per cent solution of honey, sugar, protinex were placed in the cages. The mating (pairing / Copulation) of the moths male and female moth was also observed in the laboratory Chimney cages and duration and mode of copulation was also studied. The data on oviposition by female moth, eggs and their development and daily changes in eggs were recorded Pre-oviposition, oviposition and post-oviposition period: The time taken by a female moth after mating and start of egg laying was taken as the pre-oviposition period. The number of days for which a female continued to lay eggs was considered as ovi-position period. During oviposition period, number of eggs per female was counted daily. On the basis of number of eggs laid per day throughout oviposition period, total number of eggs laid by a female was worked out by adding the number of eggs laid on different days. The time between termination of egg laying and death of the female was taken as post-oviposition period. The longevity of mated as well as unmated moths was recorded. To record observations on adult morphology, freshly emerged moths were killed in ordinary killing bottle and stretched. For the study of the effect of different host species (food plants) on the fecundity, larvae and pupae period and longevity of adults, the experiments laid out, for different host species were selected, tested and observations were recorded.

11 3.6 Effect of Temperature and Relative Humidity on Insect Biology: Required temperatures were maintained in B.O.D. incubators. Relative humidity (RH) was maintained by adding distilled water into concentrated sulphuric acid (specific gravity 1.84) by adopting the method suggested by Buxton (1931) and Buxton and Mellanby (1934). To maintain different humidity conditions, equal volume of concentrated sulphuric acid and distilled water were mixed to get stock solution. It was found that 100 ml of acid ml of water gave ml of stock solution. Required relative humidity from 40 to 80 per cent was maintained by adding stock solution and distilled water in the following quantities: Stock Solution (ml) Water (ml) Relative Humidity (%) Different concentrations of the acid were kept on the bottom of desiccators and a porcelain plate was used so as to keep the rearing beakers above the acid solution. The lids of the desiccators were tightly closed by grease. Then, the desiccators were kept in the B.O.D. incubators set with required temperature. The effect of temperature and humidity on different stages on insect pests were studied. 3.7 Morphological and Biometric study: For morphological studies adults preserved specimens were treated with 10 per cent KOH for 24 hours to soften the hard chitinous parts of the body of the moth. Traces of KOH were later removed with water and 10 per cent acetic acid. Specimens thus treated, were kept in clove oil. Temporary mounts of appendages (foreleg, midleg, hindleg, antennae), and wings were prepared in the glycerine. For permanent mount different parts of the moth were passed through ascending grades of Alchhol (10, 20, 50, 70, 90 per cent and absolute alcohol) and then mixed with Nyline or Xyline and finally

12 Plate- 4 Stereo zoom microscope

13 mounted in Canada balsam. Length and width of the eggs was measured with the help of Stereo Zoom Research Microscope and stage micrometer. For morphological studies of larvae, all the instars were preserved in 70 per cent alcohol. The later instars larvae were boiled in water for 1-2 minutes before preservation. The morphometric (biometric) observations were taken with the help of Stereo zoom research microscope (Plate- 4), ocular micrometer and stage micrometer for measuring small body parts. The body structure was also studied and the diagrams were drawn with the help of camara lucida. The length, width and head size of different larva instars were measured with the help of ocular micrometer / stage micrometer. For the morphological study of pupa, the length, width and other morphological characters of the pupa were also studied. 3.8 Population Study: Population study was done by two methods 1. The insect population in natural stands was studied by random sampling method (Snedecor and Cochran, 1967 and Southwood, 1978). The experimental sites and plots were selected in sal and rohini forest at different locations i.e. in Timli and Barkot, and on toon plants in outdoor cages of Insectory. Plants selected were marked for collection of insect. In each site three plots of 0.5 hectare were laid out. In one plot 25 per cent trees were assessed for monitoring pest population. In each plant, population data related to number of larvae per selected branch (from tip to base) were recorded. Thorough search for larvae was carried out in the sample twigs. The first and second instar larvae, were also carefully counted. 2. The mature larvae of the H.talaca pupate in soil under humus. The pupal population was monitored and studied by specialized technique (Singh, et al., 1989). For that eight pits of 30cm 3 (in FRI) and 100 cm 100 cm 30 cm (in Timli and Barkot) were dug out around the tree base in randomly selected trees. Four pits were dug near the base of plant in a circular manner and four on the outer periphery of the circle (Plate- 5 A and B). The number of pupae collected per pit was counted and observations were recorded at monthly interval from March 2007 to November 2009 and data were

14 compiled. The decrease and increase of the pupal population in the field indicated fluctuation in population level was studied at monthly interval. The variation in the population in different months of the year was studied to ascertain maximum and minimum level and peak period of activity. The impact of various factors responsible for population fluctuation in relation to the changing climatic condition mainly temperature, humidity and rainfall was recorded. The meteorological data were obtained from Range offices of every sites, during the period of study Host Range and Food Preference: The food preference experiments were conducted under the laboratory conditions. Out of 25 host plants, Toona ciliata, Delbergia sissoo, Malotus philipinsis, Cassia glouca and Murraya koenigii were selected for study of host preference under laboratory conditions. These hosts were short-listed on the basis of field observations where larvae of this pest were found to feed on these species. Experiments of food preference were conducted by two different methods. 1. In each set of experiment with different food plants 10 larvae of H. talaca (Ist instar / newly hatched) were released in chimney cage supplied with fresh food from selected host species. The leaves were changed after 24 hours and the leaf area consumed was measured with the help of leaf area meter. The fresh leaves were changed daily and the same larvae were released for further feeding. The observations were taken for each larval stage (Plate- 5 C). 2. Selected four host plants (potted plants) were placed in wooden cage. One pair of mated moth was released in the cage and observation on egg laying on every host plant was taken every day. Plants were replaced by one another in the cage to avoid effect of light in every direction of plants (Plate- 5 D) Nature and Extent of Damage: Nature of damage: The nature of damage due to H. talaca was studied both under laboratory and field conditions. Feeding habits of larvae of different instars were observed during April

15 (A) (B) (C) (D) Plate- 5 (A) Population Study of pupae in Barkot Forest. (B) Population Study of pupae in Timli Forest. (C) Leaf area meter used for food preference. (D) Multiple choice for egg laying of moth.

16 2007 to June To study the nature of damage in laboratory conditions, 15 neonate larvae were taken and released singly on sprouted leaves of potted plants placed in wooden glass cages. Feeding behavior of different larval instars was then observed and the observations on the damage caused to the leaves and symptoms produced by each instar were recorded Extent of damage: To assess the damage potential in the laboratory, 15 neonate larvae were released singly in glass tubes, the mouth of which were closed with muslin cloth, tied with rubber band. Fresh twigs with leaves were weighed and provided to each larva for feeding. After 24 hours, unused food in each glass tube was removed and weighed. Freshly weighed twigs were provided daily to repeat the observations. The quantity of food material consumed and damaged caused by a particular larval instar was noticed. The extent of damage was recorded in the Barkot forest range and in outdoor cages of Insectory. Trees of different age classes were marked to assess the damage caused by the pest. Three branches were randomly selected and removed from the lower, middle and upper portion of the trees. The leaves were removed and weighed. Extent of damage in the field (Barkot and outdoor cages of Insectory) was calculated as per the standard method prescribe in Disease and Insect Survey Manual (Bakshi, 1977). It is estimated by counting the number of leaves in the sampled twigs in all the four directions and in all three Strata. Code Damage (% Defoliation) Degree 0 No damage/ > 1% damage None % Light (L) % Moderate (M) % Heavy (H) % Severe (S) The per cent defoliation was calculated by counting the number of insects in the sample twigs, total number of leaves in the twigs and number of leaves damaged by them. However, a modification was done in the methodology i.e. code 0 include the

17 percent defoliation less than one in No damage category and the degree was considered as None. Total the foliage loss was assessed by the following formula (Singh, 2005). (L W + M W + T W) Foliage Loss = 3 Where, L W = Weight of leaves from the lower branch M W= Weight of leaves from the middle branch T W = Weight of leaves from the top branch Defoliation symptoms appearing on the twigs/ individuals trees the forest were also recorded by visual observations during March March Control: Natural enemies: Larvae and pupae of H. talaca infested with various parasitoids were brought to the laboratory and Parasite were reared and identified by the taxonomist of the Division of Entomology Biopesticide testing: For eco- friendly control of target pest, six commercially available biopesticides namely Bt. (Biolep) (Serotype H-3a, 3b, Strain Z-52. The bacterium is aerobic, gram positive, rod shaped, spore former belonging to family Bacillaceae), Agro Bt. (Jatropha extract-40%, Pongamia extract-15%, Castor extract-15%, Curcuma longa- 15%, Chitinase enzymes-10%, Dissolving agent-10%.), Trasco (Azadirachtin-1%, Solvent- 54%, Neem oil- 30%, Emulsifier-15%), Floraguard (Natural Alkaloids-14%, Natural Lactones-03%, Aqueous Media-83%), Neem oil (Extracted from seed kernals, 100%) and Neem cake (Deoiled neem seed cake) were selected.

18 Bio-efficacy of commercially available biopesticides The relative toxicity of various biopesticides against third instar larvae of H. talaca was investigated using Potter s Tower (Plate- 6 A). The third instar larvae were separated out and kept in glass beakers. They were starved for about 12 hours; ten starved third instar larvae were then transferred to glass petridish and sprayed under the spray tower. In all, 3 replications of a treatment were made. The desired concentrations of each commercially available biopesticides were prepared in the laboratory by diluting them with distilled water. The measured quantity i.e. 2 ml of each water emulsion or solution of biopesticides were then uniformly sprayed by the spray tower based on the principle of Potter s Tower at the pressure of 1.09 kg/cm 2 area. The sprayed larvae were then transferred to clean, marked, glass beakers, or plastic beakers of 1000 ml. capacity, lined at the bottom with filter paper. Mouth of such beaker was then covered with muslin cloth tied over by an elastic rubber band. Fresh untreated host leaves were provided as food to the larvae. The beakers were kept in the laboratory conditions at C temperature and 65-70% relative humidity. Observation on the dead and moribund larvae were taken. Such larvae are removed and counted. Observation was taken after every hour of treatment. The average mortality in each concentration was calculated. The percentage of larvae killed from 6 to 72 hours and obtained data were subjected to probit analysis and nested anova (Finney, 1971: Busvine, 1971). The relative toxicities of various insecticides tested were calculated based on their LC 50 and LC 90 values and obtained data were subjected to probit analysis and nested anova using software package SPSS and GENSTAT Residual toxicity of commercially available Biopesticides: For determining the residual toxicity of selected biopesticides, experiments were conducted in outdoor cages. In all, three trials were laid out. The potted host plants or planted host plants in outdoor cages were sprayed separately with different concentrations of available biopesticides with the help of Automizer and Foot pump (Plate- 6 B & C). Each concentration was replicate three times. In each replication, ten

19 larvae were used. The untreated control was kept for all the experiments. The observation on dead and moribund larvae were taken from 6 hour to 15 days. The field trials were conducted during peak period of activity. Sampling of host plant was done randomly and sampled plants were tagged for different treatments. During sampling each plant/ tree was divided into lower, middle and upper strata. A baseline was selected and plant spacing was maintained during the sampling and tagging. A row was left as a buffer. Pretreatment observations with respect to numbers of larvae of H.talaca per sample were taken before applying the treatments. Seven different concentrations of selected biopesticides were selected and all treatments were sprayed on the plants with the help of a foot pump. All treatments were repeated three times. Control experiment was also kept by observing some sampled plants without any treatment. The experimental design followed was Randomized Block Design (RBD), where host plants were selected by random sampling in three replicates of each treatment. Seven treatments were applied besides the control. The treatments were applied to the plants randomly. The experimental layout for conducting experimental is presented below: Codes of Treatments Name of treatments Concentration (%) T1 Biolep 2.0 T2 Biolep 1.0 T3 Biolep 0.5 T4 Biolep 0.25 T5 Biolep 0.13 T6 Biolep 0.06 T7 Biolep 0.03 T8 Biolep water Replication 1 Replication 2 Replication 3 T2 T3 T4 T7 T5 T6 T4 T7 T2 T3 T1 T5 T5 T6 T3 T1 T4 T7 T6 T2 T1 Experimental layout Field layout

20 Plate- 6 (A) Spray of biopesticides by Potter s Tower. (B) Field application of biopesticides. (C) Automizer used for spraying on potted plants.