MATERIALS AND METHOD

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Bertram Richard
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1 Chapter - 3 Histomorphology, Ecology and Biochemistry of leaf galls of Ficus glomerata Roxb. induced by Pauropsylla depressa Crawford. MATERIALS AND METHOD Field observations were confined in Saharanpur district and adjacent areas, where the main host plants of Ficus glomerata Roxb. are found in good number (Fig.-11). Following materials and methods were employed for different studies:- (A) For gall formation: - For noting gall formation, a healthy twig of F. glomerata was selected having young leaves. These leaves were covered by fine muslin cloth and inside it a freshly mated Pauropsylla depressa female was released for egg laying (Plate - 11A). After oviposition the female was removed. Now, at regular interval gall size and colour was noted till the emergence of mature 5 th nymphal instar from the gall through lacerated opening. The shape and size of gall was noted under binocular microscope. Healthy and gall infested leaves of F. glomerata were also collected from different localities of Saharanpur district and were fixed in different fixatives for histomorphological studies. (B) For histomorphological studies: - Morphology of the gallinaceous and healthy leaves were carried out by observing external feature, internal structure, size variation and shape etc. of different phages of gall, i.e., young, mature and old gall under binocular microscope. Structure of monolocular and multilocular 13

2 gall was seen by plucking the infested leaves of Ficus glomerata having all stages of the gall. For anatomical observations samples of leaves with galls, in successive stages of development, were fixed in FAA (Formalin, acetic acid and 50% ethyl alcohol, 1:1:18, v\v), dehydrated with ethanol series and embedded in paraffin wax. Transverse and longitudinal serial sections were cut with a rotary microtome of 6, 8, 10 and 12mm thickness. The histological sections were stained with safranin and fast green and fuchsin and astra blue. Hand cut sections of the living material were also used for a few observations. Heidenhain s ironalum haematoxylin together with orange G proved to be the most satisfactory stain for general use, though, safranin and gentian violet were also used in staining some of the sections. (C) For ecological studies:- For this purpose various surveys were carried out in the different areas of Saharanpur district and adjacent areas, viz, Saharanpur proper, Puwarka, Gangoh, Sarsawa, Nakur, Behat, Remound depot, Shakumbhari Davi and Kalsia etc (Fig - 11). Infestation percentage was recorded using following formula. Infestation % = No. of plants found infested Total no. of plants observed Effect of different ecological parameters, viz., temperature and relative humidity was recorded by using temperature and 14

3 humidity control cabinet (Plate 11B). For this purpose a specific number of P. depressa adults were kept in hurricane glass lantern chimneys, having fresh leaves of the host plant (Plate - 6). These chimneys were covered at top by fine muslin cloth and bottom was placed in a petridish. Such chimneys were placed in the temperature and humidity control cabinet. These were subjected to different temperature levels, viz, and R.H. levels, viz., 0, 20, 40, 60, 80, 100. Following formula was used for recording intensity of gall formation. No. of gall infested leaves Intensity of gall formation = 100 Total no. of leaves examined Seasonal cycle was studied in Saharanpur district and adjacent area, where the main host plants of F. glomerata Roxb. are found in good number. Different plants were randomly selected from the field area. Infested leaves were also plucked randomly from the plants in different months and infestation percentage was noted. Number of adults was also calculated per five sweeps using insect collecting hand net during different months. Temperature and humidity data were also recorded using field thermometer and dial hygrometer (Plate 10C). (D) Effect of galls on the host plant: - Some plants were selected in the field area and noted the infestation percentage and effect of galls on the general health of the plant. 15

4 (E) For biochemical studies: - For these studies, young, mature and old gall, healthy and diseased young ( cm.) and old ( cm.) leaves of almost equal size were collected form F. glomerata plant. The leaves were carefully washed in running tap water and dried in four layers of filter paper (Plate 7 C). Leaf and gall tissues of similar size (1-2 mm and 4-5 mm 300 mg. each) were carefully removed by scalpel and homogenized in chilled phosphate buffer (10 ml ph 6.1). Homogenate was centrifuged at 5000 rpm. in a centrifuge (Plate 10 A). The supernatant was used for the determination of free amino acids, total proteins, total phenols, total sugar and some supernatant (kept at 0º) was used for enzyme activity. Fresh as well as dried healthy and infested leaves (gall tissue of young, mature and old gall) were used for various biochemical estimations: - (1) Estimation of free amino acid: - The amino acids were extracted and estimated by the procedures given by Porter, et.al., (1957), and Block, (1963). The samples were run in n- butanol:acetic acid:water (74:1:19:2) in the other dimension. Chromatograms were developed at 100ºc after spraying with 0.5 % ninhydrin in acetone. All the chromatograms were run in duplicate and for each set of unknown, a standard mixture of known amino acids was also run. The ninhydrin positive spots were eluted in 4ml of 75% ethanol containg 0.05% CuSo 4. 5H 2 O and read in colorimeter at 540 nm (Plate 9 B). The concentration of different amino acids were expressed as glycine equivalent. 16

5 (2) Estimation of total protein: - Total proteins were estimated by the procedure of Lowry, et. al. (1951) using bovine serum albumin as the standard (Plate 8). (3) Estimation of total phenols: - Total phenolic substrances were estimated by employing Folin Denis reagent as described by Bhatia, et. al, (1972) and were calculated from a standard curve earlier prepared using tannic acid. (4) Estimation of reducing sugar: - Estimation of reducing sugar was done by the method of Miller, (1972). For this purpose, 500 mg plant material was treated with 10.0 ml of 80% ethyl alcohol. In 3.0 ml of alcoholic extract, 3.0 ml of DNSA (3, 5- dinitrosalicylic acid) reagent was added. The mixture was heated for 5 minutes in a boiling water bath. After the colour had developed, 1.0 ml of 40% Rochelle salt was added when the contents of the tubes were still warm. The tubes were cooled under running tap water (Plate - 7C). Absorbance was recorded using colorimeter at 515 nm. The amount of reducing sugar was calculated using standard curve prepared from glucose. The quantity of reducing sugar was expressed as mg\g fresh weight of tissue. (5) Estimation of total soluble sugar: - The amount of total soluble sugars was estimated by phenol sulphuric acid reagent method (Dubios et. al., 1951). 500 mg each of fresh normal and galled plant material was homogenized with 10 ml of 80% ethanol. Each sample was centrifuged at 2000 rpm for 20 minutes (Plate 10A). The supernatant were collected separately 17

6 to 1.0 ml of alcoholic extract. 1.0 ml of 5% phenol solution was added and mixed. Then, 5.0 ml of 96% sulphuric acid was added rapidly. Each tube was gently agitated during the addition of the acid and then allowed to stand in a water bath at 26-30ºc for 20 minutes. The O.D of the characteristic yellow orange colour thus developed was measured at 490 nm in a colorimeter after setting for 100% transmission against the blank. Standard curve was prepared by sing known concentration of glucose. The quantity of sugar was expressed as mg\g fresh weight of tissue (Plate 7 A, B). (6) Estimation of ph changes: - ph changes were determined by ph measurement strip. B.D.H. p.h papers were used (Plate 10, B). Well developed galls produced by P. depressa on F. glomerata were collected and carefully opened without injuring the nymphs. The nymphs of P. depressa were washed with distilled water and the ph of the solution obtained was tested by the Clark, (1920) color indicators as well as ph papers. (7) Estimation of enzymatic changes: - IAA and IAA oxidase activity in fresh samples was determined by the procedures of Schneider et.al. (1972) and Rabin and Klein (1957) as described by Witham et. al. (1971). Leaf and gall tissues of similar size (1-2 mm and 4-5 mm, 300 mg each) were carefully removed by scalpel and homogenized in chilled phosphate buffer (10 ml, ph. 6.1). Homogenate was centrifuged (5000 rpm) in an ultra centrifuge (Plate 10, A). The supernatant (kept at 0ºc) was used for enzyme activity. 18

HiPer Carbohydrates Estimation Teaching Kit (Quantitative)

HiPer Carbohydrates Estimation Teaching Kit (Quantitative) Product Code: HTBC003 Number of experiments that can be performed: 10 Duration of Experiment Protocol DNSA Method :1 hour Phenol Sulphuric Acid