ChZnNF
Bulk Zinc oxide (5 g) was dissolved in hot distilled water and stirred using magnetic stirred until completely dissolved. Water soluble chitosan (1 per cent) was filtered and added to the zinc oxide solution and stirred continuously for 48 hours and subjected to ultrasonication to obtain the nanoformulation
(Vinay et al., 2016).
PfZnNF
Fresh broth culture of
P. fluorescens was centrifuged to obtain the microbial extract. The bulk zinc oxide (5 g) was added to heated distilled water (850 ml) and stirred well. To this zinc oxide mixture and the microbial extract (150 ml) was added and stirred for 24 hours. The final solution was subjected to ultrasonication to reduce the particles to nano size
(Vinay et al., 2016).
PASNF and PAAgNF
Pomegranate aril obtained from fruits was sun dried and crushed in sterile pestle and mortar. The extract was filtered in Whattman 5 filter paper to remove impurities. This filtrate was used for the synthesis of sulphur and silver nanoformulation (
Srikanth, 2018).
Sodium thiosulphate (3 g) was used as precursor for sulphur, which was added to 100 ml of 18 per cent of pomegranate aril filtrate and stirred well using magnetic stirrer. Further, ml of 20 per cent citric acid was added to the above mixture and stirred for 24 hours. The final solution was then subjected to ultrasonication to produce nanoparticles (PASNF).
For PAAgNF, silver nitrate (3 g), was added to 490 ml of distilled water and stirred well before boiling in microwave oven. Later, was stirred for 24 hours and subjected to ultrasonication for 30 minutes.
Characterization of nanoformulation
ChZnNF, PfZnNF, PASNF and PAAgNF were subjected to Particle Size Analyser (PSA) to determine the size of the nanoparticle in each of the formulation synthesised. Scanning Electron Microscope (SEM) (Carl Zeiss-EVO-18-UK) with Energy Dispersive X-Ray Spectroscopy (EDS was used to analyze the surface topology and particle morphology images of the nanoparticle in the nanoformulation. Besides,the element present in the nanoformulation was discerned in Energy Dispersive X-Ray Analysis (EDAX).
Evaluation of nanoformulation against C. truncatum by in vitro assay
In order to evaluate the affectivity of the nanoformulation,
in vitro assay was carried out using the standard poison food technique (
Shravelle, 1961) against
C. truncatum using sterilized Potato Carrot Agar medium. The concentrations of each formulation are as discussed in Table 1-5.
Control was maintained by using PCA medium without incorporation of nanoformulation. Radial mycelial growth of the test fungus in treated and control treatments was recorded. Further, percent inhibition was calculated by using the formula given by
Vincent (1947).
Where,
C= Radial growth of mycelium in unamended medium (Control).
T= Radial growth of mycelium in amended medium.
Evaluation of nano formulation against C. truncatum under glasshouse condition
The effective concentration from each of the nano formulation was subjected to evaluation against
C. truncatum under glass house pot experiment. Seeds of soybean (JS-355 and DSb21 varieties) were treated accordingly as explained in Table 5 and sown in pots filled with sterile pot mixture. Spraying of respective treatment was carried out at 30 and 40 days after sowing. The seedlings were challenged with the test fungus by artificial inoculation technique. Observations were recorded from the first day symptom appearance; Percent disease index was calculated at three days interval and FESEM analysis.
Phytotoxic effect of nano formulation on soybean plants
Phytotoxicity of nano formulation was assessed by spraying plants with various concentrationsat 1, 3, 5, 7, 9, 11, 13 and 15 days after the spray.