Plant material and collection of leaf samples
African marigold plants (133-1-2-Lemon red germplasm) (Fig 1a) were sown for summer (January, 2018) and winter (August, 2018) seasons under screen house and field conditions and the crop was monitored till harvesting season in May, 2018 and March, 2019 for summer and winter season crops respectively. When the leaves were naturally infested with
T. urticae, leaf samples (infested and uninfested) were collected on a suitable day in each season and were brought to acarology laboratory, Dept. of Zoology, CCS HAU, Hisar. Two leaves each from tender, grown up and older leaf stage were collected. Mixed population of egg (Fig 1b), larva, nymph (Fig 1c) and adult (Fig 1d) was calculated from both adaxial and abaxial surface of leaves under stereo zoom binocular microscope. Average number of mites per leaf were correlated with mineral, phenol and proline content.
Estimation of minerals
Nitrogen, phosphorous and potassium content in marigold leaves was estimated after feeding by
Tetranychus urticae in separate sets, to ascertain the effect of mite feeding on these parameters during summer and winter season. These were compared with non infested leaves acting as control for each set.
1.1 Digestion of sample for nitrogen estimation
Under replicated conditions we took powdered material (100 mg) into a 100 ml conical flask and 10 ml diacid mixture (Sulphuric acid + Perchloric acid; 4:1) was added to each of the flask (Fiske and Row, 1925). These flasks were covered with watch glasses and allowed to stand overnight
(Fiske and Row, 1925). Afterwards, to dissolve the solid particles, heating was done and a clear colourless solution was obtained.
Then one ml of HCl concentrate was added to each sample and again heated so as to get a colourless solution. Allowed the solutions to cool down and made the final volume to 25 ml with 1 per cent HCl in distilled water. Simultaneously, a blank was also run down without the sample. The thorough mixing of solutions was done and then used for analysis of nitrogen.
1.2 Nitrogen estimation
Nitrogen content of leaf was estimated by using the method proposed by
Lindner (1944). Into a 50 ml volumetric flask, we took 10 ml aliquot (digested material) and added 2.5 N sodium hydroxide (2 ml), percent sodium silicate solution (1 ml). This neutralized the excess acid and prevented turbidity. About 5 ml aliquot of this solution was taken in a 10 ml graduated test tube and 0.5 ml of Nessler’s reagent was added to it. The test tubes were then allowed to stand for 5 min so that maximum colour develops. On UV-vis spectrophotometer, at 525 nm OD of the solution was recorded. Compared the reading for each sample with the standard calibration curve of ammonium sulphate to examine the per cent nitrogen content in marigold leaves.
2. Phosphorus estimation
Method of Jackson (1973) was used to estimate the phosphorus content of leaves. We took a 25 ml volumetric flask and added one ml of aliquot into it. Afterwards, it was diluted by adding 9 ml distilled water. Then one ml of it was taken in a separate test-tube and added 3.2 ml of distilled water, 0.2 ml 10N H
2SO
4 and 0.4 ml of 2.5 per cent ammonium molybdate into it. Mixed it properly and added 0.2 ml of 1, 2, 4 amino naphthol sulphuric acid reagent (freshly prepared) into it. Using UV-vis Spectrophotometer reading was taken at 660 nm.
3. Potassium estimation
From the digested sample direct reading
(Richards, 1954) were taken using Micro Flame Photometer (Elico CL 361, India) for the estimation of potassium. Fed the instrument with five ml of sample and for each sample observations were recorded. Standard KCl for potassium was used to note the potassium content.
4. Estimation of total phenols
4.1 Extraction
Marigold leaves were powdered for each sample (500 mg) and 80 per cent methanol (30 ml) was added to them separately in a 100 ml volumetric flask. Then flasks with samples were placed in water bath (60°C) for one hour. Afterwards, allowed the flasks to cool down and filtered the contents using Whatman filter paper No. 1. Collected the supernatant in 100 ml beakers and using 80% methanol solution, final volume was made 30 ml. The contents were mixed properly. A blank was prepared simultaneously but without the sample.
4.2 Total phenol
The extracted content was used for total phenol estimation of uninfested and infested leaves
(Swains and Hillis, 1959). 0.5 ml methanolic extract and 7.5 ml distilled water was taken in a test tube and 1N of Folin Ciocalteu‘s Phenol (1 ml) was added to it. It was kept for few minutes and one ml of saturated Na
2CO
3 solution was added to it. The solution was allowed to stand for one hour till the blue colour developed. The absorbance was recorded at 725 nm using UV spectrophotometer. A blank was prepared using same procedure but without extract in it. A standard curve was prepared with gallic acid and total phenol content was determined.
5. Estimation of proline
Estimation of free proline content was done following method of
Bates et al., (1973). We took fresh leaf samples (500 mg) and homogenized them. Added 5 ml of 3 per cent sulphosalicylic acid to it and centrifuged at 5,000 rpm for 15 min. The supernatant was extracted. The supernatant (2 ml) was mixed with 2 ml of acetic acid and 2 ml of acid ninhydrin. The contents were boiled for 30 minutes and then allowed them to cool down. UV spectrophotometer was used to note the absorbance at 520 nm and toluene was taken as blank. Graded concentrations of proline in 3 per cent sulphosalicylic acid was used to prepare a standard curve. Proline content was expressed as μg/100 mg fresh weight of the leaf tissue. During summer and winter season, per cent loss in photosynthetic pigments, total sugars, non reducing sugars and mineral contents were calculated under screen house and field conditions by following formula:
Per cent increase in total phenols and proline was calculated by following formula during summer and winter season in screen house and field experiments:
Statistical analysis
‘OPSTAT’, a standard statistical analysis tool was used for the data analysis. To see the effect of observation place, season and leaf condition on the population dynamics of
T. urticae, critical difference was calculated by applying ANOVA (Analysis of Variance) through two factorial completely randomized design (CRD) and the statistical significance of data was assessed. Values with same superscript were at par with each other. Correlation coefficient ‘r’ was also calculated to see the effect of mite incidence on the biochemical parameters. Correlation variables vary together and are defined by:
Where,
X = Number of mites.
= Mean number of mites.
Y = Amount of mineral content/ phenol/proline.
= Mean of mineral content/ phenol/proline.
