Sample collection
The samples were collected from a nearby area of agricultural land
i.e. Sardar Vallabhai Patel University of Agriculture And Technology, Meerut in the year 2020. We use only fresh samples of two weeds for further work.
Extracts preparations
Before the extract preparation, we wash the samples, shade them dry for 24 to 72 hours, chopped and macerated. When samples were completely dry, we took the sample and organic solvent at a particular ratio (1:10) in soxhlet apparatus for extract preparations.
Culturing of pathogens
Fungal phytopathogens were isolated from the infected Bengal gram (
Cicer arietinum) crop as
Fusarium oxysporum,
Rhizoctonia solani and
Sclerotium rolfsi. These fungal phytopathogens were isolated on a PDA medium by pure culture technique methods. We took the infected part of Bengal gram as root, stem and leaf and put it on a basic fungal culture medium
i.e. potato dextrose agar and after that, all the Petri plates were incubated at culturing temperature
i.e. 28°C for 24 hours. After that growth appeared in all the plates. Based on basic morphology we isolate the fungus and inoculate the fungal culture into a fresh Petri plate of PDA for further identification.
Antioxidant activity of weed extracts by different methods
DPPH methods
DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical is an antioxidant in an electron-transfer-based process that produces a violet way out in ethanol. This free radical, firm at room temperature, is compact in the existence of an antioxidant molecule, giving rise to a colorless ethanol solution. Uses of the DPPH assay provide an effortless way to estimate antioxidants by spectrophotometer, so it can be constructive to assess various goods at a time. The antioxidant activity percentage (AA %) of each substance was assessed by DPPH free radical assay. DPPH radical measurement scavenging activity was performed as described by methodology Brand-Williams. The samples were reacted with the stable DPPH radical in an ethanol solution. The reaction mixture consisted of adding 0.5 ml of sample, 3 ml of absolute ethanol, 0.3 ml of DPPH radical solution and 0.5 mm of ethanol. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The color turn (from deep violet to light yellow) was recorded at 517 nm Absorbance after 100 min of reaction using a UV-VIS spectrophotometer
(Brand-Williams et al., 1995).
FRAP methods
The ferric-reducing ability of studied plant materials was assessed following the method described by
Benzie and Strain (1996). The final results were articulated and have ferric reducing ability equivalent to that of 1 mm FeSO
4, particularly expressed as mol Fe (II) equivalent/g sample in dry weight.
ABTS methods
Free radical scavenging activity of plant samples was determined by ABTS radical cation decolorization assay
(Pellegrini et al., 1999). The ABTS·+ cation radical was produced by the reaction between 7 mm ABTS in water and 2.45 mm potassium persulfate (1:1), stored in the dark at room temperature for 12-16 h before use. The ABTS·+ solution was then diluted with methanol to obtain an absorbance of 0.700 at 734 nm. After the addition of 5 μl of plant extract to 3.995 ml of diluted ABTS·+ solution, the absorbance was measured at 30 min after the initial mixing. An appropriate solvent blank was run in each assay.
In vitro testing of isolated fungal phytopathogens on legume crop Bengal gram (Cicer arietinum)
Seeds of Bengal gram were obtained from the local market of Meerut. They were sown in pots of equal size. Each pot had 100 gm of soil. Each treatment was replicated three times with 10 seeds per pot. Pots were arranged in a completely randomized design (CRD) and were maintained at field capacity in a uniform open environment. One disc of all the fungal phytopathogens (
Fusarium oxysporum, Rhizoctonia solani and
Sclerotium rolfsi) cultures was added to each pot and then irrigation was done with tap water. Five healthy seedlings were left in each pot. Thereafter, pots were irrigated with different concentrations (5%, 10% and 15%) of all solvents of both
Dactyloctenium aegyptium and
Chenopodium album root extracts (original stock solution) by diluting it with the appropriate amount of distilled water. Control pots were irrigated with tap water. At the time of maturity of the Bengal gram crop, data were collected based on different growth parameters, such as germination % disease symptoms in leaves, stems and roots.
In vivo testing of pathogens on culture media
This testing is done using food poisoning techniques. It is a very effortless and normal method to resolve the inhibition zone. The weed extracts were used beside the fungal phytopathogens
Fusarium oxysporum, Rhizoctonia solani and
Sclerotium rolfsi. One gm crude samples of both weeds diluted with 10 ml of each solvent
i.e. Ethyl acetate, butyl alcohol, benzene, methanol and sterile water and from this suspension, 1250 μl, 2500 μl and 3750 μl is added to the PDA melted medium to obtain the desired concentration to form a volume of 25 ml of suspension was poured into each sterile Petri plate but in controlled Petri plate free from extracts concentration and allowed to be stabilized. Three duplicates (plates) were utilized for each treatment. For test fungal cultures, each plate was infected with a 6 mm diameter disc and incubated at 28°C until the control plates reached maximal growth.
Statistical analysis
At maturity, data on different extracts at different concentrations were recorded and statistically analyzed using a weighted mean.
The weighted mean was applied in the present study. The following mean was used for the study:
Weighted mean
Where:
Xi = Worth of a thing.
Wi = Item’s weight.
