First Report on Synthesis of Green Nanoparticles and Their Bio-Efficacy against Colletotrichum truncatum Causing Pod Blight Disease in Soybean

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.

Characterization of synthesized green nano formulation
 
The color of the end product of ChZnNF and PASNF was off white precipitate, PfZnNF was white to yellow precipitate and PAAgNF was chocolate brown to black respectively (Fig 1). The mean diameter recorded for ChZnNF, PfZnNF, PASNF and PAAgNF using Particle Size Analyser was 73.6 nm, 9.9 nm, 79.8 nm and 83.5 nm respectively as indicated in Fig 2 -5.The SEM analysis with EDS revealed that zinc element was present in ChZnNF and PfZnNF, which was rod shaped (Fig 2) and rod to irregular shaped (Fig 3) respectively. Sulphur was detected in PASNF and was spherical to irregular in shape (Fig 4), while silver nanoparticle was detected in PAAgNF, which was spherical in shape (Fig 5).
 

 

 

 

 

       
Similar results were recorded by Vinay et al., (2016) who synthesized green nanoformulations i.e. Pseudomonas fluorescens and chitosan based zinc nanoformulation. The findings are in similarity with the results of various researchers who used chitosan in synthesis of zinc oxide nanoparticles (Jae-Wook et al., 2019).
       
Various researchers Kouzegaran and Farhadi (2017), Tripathi et al., (2018), Ragab and Saad-Allah (2020) synthesized sulphur nanoparticles through green approaches by using plant extracts. Characterization of these nanoparticles revealed that the size was ranging from 20 to 120 nm and shapes were ranging from spherical to random shape. Pomegranate peel as reducing agent was used in nanoparticle synthesis by Phongtongpasuk and Poadang (2015) and Upadhyay (2018).
 
In vitro assay of nanoformulation against C. truncatum
 
The observation recorded on percent mycelial inhibition for respective treatments are represented in Table 1-4. ChZnNF at its highest concentration (@1500ppm) showed 49 per cent inhibition, while PfZnNF (@ 1500 ppm) showed inhibition of 58.17 per cent which was comparatively higher than ChZnNF as indicated in Table 1 and 2 and depicted in Fig 6 and 7. In case of PASNF, highest inhibition of 27.06 per cent at 2000 ppm concentration (Table 3) was noted. Whereas, PAAgNF showed an inhibition of 84.71 percent at 500 ppm (Table 4). Thus, among all the green nanoformulations, PAAgNF was found to be effective in inhibiting the mycelial growth of C. truncatum (Fig 8 and 9).
 

 

 

 

       
The results were in conformity with the results of Lamsal et al., (2011), Rosa-Garcia et al., (2018) and Srikanth (2018) against Colletotrichum spp.
 
Evaluation of nanoformulations on soybean challenge inoculated with Colletotrichum truncatum under glasshouse condition
 
Among 16 treatments (Table 5), silver nitrate at 500 ppm concentration did not record any disease symptoms after second spray in the two varieties. Pomegranate aril based silver nanoformulations (PAAgNF) did not record any anthracnose symptoms on JS 335 variety 15 days after second spray whereas DSb 21 recorded 0.62 disease severity. Carboxin 37.5%+Thiram 37.5% DS was on par with PAAgNF at 500 ppm concentration in case of DSb 21 variety with 1.23 PDI. In case of JS 335 variety it recorded 2.47 percent severity. JS 335 and DSb 21 sprayed with PAAgNF at 500 ppm concentration reduced 100 and 96.97 per cent anthracnose disease over control whereas Carboxin 37.5% +Thiram 37.5% DS recorded 95.74 and 96.97 per cent reduction of disease severity over control. This was followed by zinc oxide at 1500 ppm concentration with 3.70 and 8.64 PDI in case of JS 335 and DSb 21 variety and sodium thiosulphate at 2000 ppm concentration with 7.41 and 4.94 PDI.The results are in conformity Srikanth (2018) and Supriya (2019) in pulse crop against fungal pathogen. Bacillus subtilis based zinc nanoparticle and chitosan based zinc nanoparticle at 1250 ppm concentration effective in managing citrus canker and bacterial wilt of tomato respectively (Vinay et al., 2016).
 
Phytotoxicity of green synthesized nanoformulation
 
It was observed under the study that ChZnNF and PfZnNF along with precursor did not show any phytotoxicity upto 2000 ppm. PASNF, sodium thiosulphate, showed toxicity with symptoms such as scorching, curling and drying of leaves. PAAgNF did not show any toxicity up to 500 ppm concentration. Toxicity to the extent of 20 per cent was observed at 15 days after spray of 1000 ppm of PAAgNF. Precursor of PAAgNF, silver nitrate showed toxicity up to 20 per cent from 10 days after spray. Toxicity symptoms observed were tip scorching and veinal chlorosis (Fig 10).
 

       
Phytotoxicity of precursor and non-phytotoxicity of the nanoparticles in case of sulphur and silver was also recorded by Supriya (2019). Similarly, non-phytotoxicity of silver nanoparticles was reported by Vanti et al., (2018).
 
Confirmation of nanoparticles on sprayed leaves
 
FESEM performed by selecting the treated leaves, showed the presence of the respective nanoparticle (Fig 11). Whereas, in the control there was no record of nanoparticle present.
 

Nanoscience in phytopathology is worthiest option as it reduces the environment pollution. There are few nano-capsules with controlled and effective release, which increases the efficiency of fungicide. Synthesis of nanoparticles through green approaches by using plant extract or beneficial microorganisms has advantages over chemical or physical method. Green synthesis of nanoparticles is simple, more convenient, requires less reaction time and is eco-friendly. In this study we evaluated four green nanoparticles, among which PAAgNF was found to be effective against C. truncatum.