Notebook | MIT-MAHE

Wet Lab Training

Wet Lab Basic Training Assessment

17^th March

All wet lab members passed the basic lab training assessment conducted by our primary investigator, Dr. Ritu Raval.
A lab familiarization session was held where we visited the lab & made an inventory of chemicals, reagents, and glassware.

Wet Lab Basic Training

Basic molecular experiments were studied, performed, and analyzed. The goal of this training session was to acclimate all the wet lab members to the lab work environment and engage in troubleshooting as and when required.

Competent cell preparation and Transformation

18^th March to 21^st May

Day 0: E. coli DH5α culture from glycerol stock was revived by plating a parent plate, which served as our source for the primary culture. All required glassware were autoclaved.

Day 1: The primary culture was kept overnight for incubation, and the acid-salt buffers were made.

Day 2: The secondary culture was inoculated. Confirmatory tests, like Indole tests and Gram staining, were carried out at every step to eliminate contamination. The competent cells were prepared, and aliquots were stored at -20°C.

Day 3: The competent cells were thawed and incubated with pET22b plasmids of 43.8 μg/mL concentration, and were transformed. The cells were then plated on ampicillin plates.

Fig A. Transformed E. coli DH5α (pET22b) plates with the control — Fig A. Transformed *E. coli* DH5α (pET22b) plates with the control.

Hence, E. coli DH5α cells were successfully made competent and transformed with pET22b.

SDS-PAGE

21^st April

Sodium dodecyl sulphate polyacrylamide agarose gel electrophoresis (SDS-PAGE) was one of the experiments performed in the basic training period to learn the skills of gel making and, familiarize with protein extraction and purification protocols.

The following gel percentages were used, given by Ms. Atheena Ma’am, who guided us through the experiment.

Table 1. SDS - PAGE gel percentages

Constituents	Resolving Gel		Stacking	Resolving Gel		Stacking
Constituents		12.5%	Stacking	15%	4%	Stacking	12.5%	15%	4%
MilliQ Water	2.303 mL	1.467 mL	3.505 mL	1.151 mL	0.733 mL	1.752 mL
30.8% Acrylamide	4.159 mL	5 mL	0.739 mL	2.079 mL	2.5 mL	0.369 mL
1 M Tris: pH-8.8	3.327 mL	3.323 mL	-	1.663 mL	1.661 mL	-
0.5 M Tris: pH-6.8	-	-	0.649 mL	-	-	0.324 mL
10% SDS	100 µL	100 µL	50 µL	50 µL	50 µL	25 µL
10% APS	100 µL	100 µL	50 µL	50 µL	50 µL	25 µL
TEMED	10 µL	10 µL	6 µL	5 µL	5 µL	3 µL
	For 2 gels			For 1 gel

The ladder was loaded in the first lane with a 10 to 245 kDa size range.
Lane 1 is protein samples lysed from E. coli DH5α.
Lane 2 is a protein of 70 kDa weight, extracted from Candida albicans, given by Ms. Atheena Ma’am.

Therefore, protein separation, purification, and analysis were successfully conducted.

Colony PCR

22^nd April

From the transformed plate, one E. coli DH5α colony was suspended in 10 µL of water. We used Takara Star 16S RNA primers for the same. The reaction mixture contents were used as follows:

Table 2. PCR Master Mix contents

Reagent	Volume (in µL)
Buffer	12.5 (2X)
DNTPs	2
Forward primer	1.5
Reverse primer	1.5
Template	1
Taq polymerase	0.25
Water	6.25

Fig C. Amplified DNA run on 1% agarose gel with ladder.

A 1kB DNA ladder was loaded into lane L.
The amplified DNA was observed as a bright band in lane 1.

Hence, colony PCR from the transformed E. coli colonies was performed, and the DNA was amplified.

Plasmid Isolation

22^nd May

From the transformed E. coli DH5α plates, one colony was resuspended in an Eppendorf tube with 30 µL of water, and the pET22b plasmid was isolated.
The QIAprep Spin Miniprep Kit – Plasmid Purification was used (QIAGEN, n.d.).
The symbols marked with a circle in the protocol were performed.

Fig D. Spectrophotometer measurement of plasmid concentration using a nanocuvette.

The pET22b plasmid used during transformation was at a concentration of 43.8 μg/mL. As observed above, 42.2 μg/mL was isolated. These readings were taken using a nanocuvette with a spectrophotometer.
The A260/280 ratio for DNA must be ideally ~1.8 to be considered pure. We obtained 1.78. The A260/230 ratio is supposed to be within 2.0 and 2.2. We obtained a lower value of 1.49, which can be attributed to phenol contamination or residual salts.
Therefore, pET22b plasmids have been successfully isolated from E. coli DH5α culture and were stored at -20°C.

References:

QIAGEN. (n.d.). QIAprep Spin Miniprep Kit | Plasmid DNA Isolation. Retrieved October 6, 2025
https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/plasmid-dna/qiaprep-spin-miniprep-kit

P. capsici Subculturing 1

+

P. capsici Subculturing 1

25^th July

Preparation of Media Plates for Subculturing

To subculture the Phytophthora capsici received from the Indian Institute of Spices Research, potato dextrose agar (PDA) and carrot agar were prepared in autoclaved glassware.

Preparation of Carrot Agar:

20 g of carrot was blended and ground using a mortar and pestle.
60 mL of distilled water was added to the carrot puree in a beaker.
The mixture was then boiled in a microwave.
The froth that developed on top was discarded to get a clear solution.
The mixture was then filtered through a muslin cloth, resulting in a clear, slightly orange solution.
The filtrate’s volume was then made up to 100 mL in a measuring flask using distilled water.
The carrot solution was combined with 1.8 g of agar in a conical flask.

Preparation of PDA:

2.4 g potato dextrose broth powder was combined with 100 mL distilled water and 1.8 g agar in a conical flask.

The prepared nutrient media were autoclaved along with the cork borer.

Fig 1. Carrot agar media and PDA before autoclaving

26^th July

Culturing P. capsici using Carrot Agar and PDA

The laminar air flow (LAF) chamber was cleaned, and the apparatus required for P. capsici culturing (disposable inoculation loops, agar plates, cork borer, etc.) was placed under the UV.
Four plates of carrot agar and PDA were made, of which three plates of each medium were designated for culturing. The cork borer technique was practiced on the additional PDA plate that was prepared.
Disks were cut from the parent culture using the cork borer, ensuring that they were not positioned too close to each other due to the disrupted hyphal network near the cut regions. The cut disks were then lifted using disposable inoculation loops.
Each disk was inverted before being placed on the agar plate to ensure that the mycelium was in contact with the agar. Some disks were difficult to remove due to the dense mycelial network.
One plate of each medium was also used to check the colony diameter from the centrally placed disk.
Two plates each of PDA and carrot agar were utilized for subcultures. Two disks were positioned at the periphery of the plates, ensuring ample separation from the edges. The procedure was conducted under the observation and guidance of the PI.
The plates were then initially placed in a cupboard, following which they were positioned in a dark incubator at 28°C, incubated upright (lid on top) to prevent the disks from losing contact with the agar.

Fig 2. Phytophthora capsici plate received from IISR — Fig 2. *Phytophthora capsici* plate received from IISR

Fig 3. Close-up picture of the mycelial plug

P. capsici Growth Curve 1 (26^th July - 1^st August)

+

Colony Diameter Measurement (Growth Curve)

27^th July (Day 1)

A divider and ruler was used to measure the diameter of the growing colony.

Plug diameter: 8 mm

Growth Curve PDA Day 1 (24 hours)

Y axis: 2.3 cm
X axis: 2.55 cm

Final reading: 1.625 cm

Growth Curve Carrot Agar Day 1 (24 hours)

Y axis: 3.6 cm
X axis: 3.6 cm

Final reading: 2.8 cm

The decontaminated cork borer was soaked in soap solution and cleaned with distilled water. It was then autoclaved for future use.

Conclusion:

The growth of the culture was observed to be more rapid on carrot agar compared to PDA.

28^th July (Day 2)

Plug diameter: 8mm

Growth Curve PDA Day 2 (48 hours)

Y axis: 4.3 cm
X axis: 4.1 cm

Final reading: 3.4 cm

Growth Curve Carrot Agar Day 2 (48 hours)

Y axis: 6.2 cm
X axis: 6.3 cm

Final reading: 5.45 cm

29^th July (Day 3)

Plug diameter: 8 mm

Growth Curve PDA Day 3 (72 hours)

Y axis: 5.95 cm
X axis: 6.1 cm

Final reading: 5.225 cm

Growth Curve Carrot Agar Day 3 (72 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

30^th July (Day 4)

Plug diameter: 8 mm

Growth Curve PDA Day 4 (96 hours)

Y axis: 7.8 cm
X axis: 7.7 cm

Final reading: 6.97 cm

Growth Curve Carrot Agar Day 4 (96 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

31^st July (Day 5)

Plug diameter: 8 mm

Growth Curve PDA Day 5 (120 hours)

Y axis: 7.8 cm
X axis: 7.7 cm

Final reading: 6.95 cm

Growth Curve Carrot Agar Day 5 (120 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

1^st August (Day 6)

Growth Curve PDA Day 6 (144 hours)

Y axis: 7.8 cm
X axis: 7.7 cm

Final reading: 6.95 cm

Growth Curve Carrot Agar Day 6 (144 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.5 cm

Lactophenol Blue Staining

+

Lactophenol Blue Staining

26^th July

Lactophenol blue staining:

A microscope slide was labelled, and a drop of lactophenol blue stain was placed at its center.
A small sample of the culture was carefully collected using a disposable inoculation loop, ensuring that the sporangium was not damaged. It was then spread onto the stain.
A cover slip was gently placed over the stained sample to prevent the formation of air bubbles.
The slide was then observed under a microscope at 10x and 40x magnifications.
Hyphae were observed under 40x magnification.

Fig 4. P. capsici hyphae observed under 40x — Fig 4. *P. capsici* hyphae observed under 40x

29^th July

Fig 5. P. capsici observed under 40x

P. capsici Water Storage

+

Water Storage for P. capsici

29^th July

P. capsici water storage:

⁠1 mL of autoclaved sterile water was added to a 2 mL Eppendorf tube.
4 fungal plugs were added to each tube.
The tubes were then parafilmed and stored at 15-28°C.

Testing Chitosan Solubility

+

Chitosan nanoparticle production

30^th July

Chitosan nanoparticle production preparation

The low molecular weight chitosan (Sigma Aldrich 448869-50G) was tested for its solubility.
Two samples of 4 mg chitosan in 1 mL of distilled water and 1% acetic acid solution were prepared. They were subjected to vortexing for 3 minutes.
The chitosan in the 1% acetic acid solution completely dissolved, whereas the water sample still had suspended chitosan particles.
To produce nanoparticles, a 2.4 mg/mL of Sodium Tripolyphosphate (TPP) solution was prepared and allowed to drop from a burette, to achieve the ideal flow rate of 60 mL/hr. The duration between two consecutive drops to achieve this was determined to be 6-7 seconds.
The volume of chitosan nanoparticles to be prepared was adjusted by determining the magnetic bead size and the container in which the nanoparticles would be prepared (30-40 mL in 100 mL beakers).

Fig 7. Chitosan in sterile water (did not dissolve)

Conclusions:

The chitosan solution had better dissolution in 1% acetic acid than in distilled water. To achieve the desired flow rate, the duration between two drops must be between 6-7 seconds.

Chitosan Nanoparticle Production 1

+

Chitosan Nanoparticle Production

31^st July

To prepare 4 mg/mL of chitosan solution, 120 mg of chitosan was added to 30 mL of 1% acetic acid solution.
The chitosan solution was subjected to stirring using a magnetic stirrer at its maximum stable speed. On observing the lack of dissolution after a few hours, it was subjected to overnight stirring.
30 mL of 2.4 mg/mL of TPP solution was prepared. The solution was passed through a 0.22 μm PVDF filter and kept ready for usage.

1^st August

30 mL of 0.4% chitosan solution was made the previous day; however, the volume had reduced to 20 mL upon overnight stirring, due to which the solution was brought up to 30 mL by the addition of 1% acetic acid solution.
The solution was then subjected to magnetic stirring. Syringe filtration was attempted; however, the process could not be performed due to the following reasons:
- Solution was highly viscous.
- A 0.22 μm pore size filter was used, which increased the risk of clogging.
After 1 hour of syringe filtration, less than 5 mL of the filtrate was obtained.
The solution was subjected to filtration using a syringe pump. This did not work since:
- The solution was too viscous.
- The volume of the solution to be filtered was very low.
On the recommendation of the PI, 1 mL of acetic acid was added to the solution and magnetic stirring was carried out.
The solution was filtered using regular filter paper, but the process was unsuccessful as the solution was absorbed into the filter paper.
As Whatman filter paper was not available, the chitosan-acetic acid solution was stored in a 50 mL Falcon tube.
30 mL of 0.1% chitosan-acetic acid solution was prepared and left for stirring overnight.

Conclusions:

Chitosan solution is highly viscous and cannot be filtered effectively using syringe filtration.

Chitosan Nanoparticle Production 2

+

Chitosan Nanoparticle Production

2^nd August

0.4% and 0.1% chitosan solutions were prepared in 1% acetic acid and stirred overnight.
The volume of both solutions was brought up to 30 mL by adding 1% acetic acid. The adjustment was necessary because the volume had decreased from 30 mL to less than 20 mL due to evaporation during stirring, as the solutions were not covered.
The 0.1% chitosan solution was filtered using filter paper. Owing to the viscosity of the 0.4% solution, an additional 1mL of acetic acid was added to it to reduce the viscosity.
The TPP solution was freshly prepared and filter sterilized using a 0.22 μm PVDF filter.
Using a burette, 6mL of TPP solution was added dropwise to 20 mL each of the 0.1% and 0.4% chitosan solutions under constant stirring at maximum stable speed. This ensured a 1:3 ratio of TPP to chitosan.
It was observed that the flow rate in the burette reduced as time passed. An attempt was made to ensure that the duration between two drops was 6–10 seconds.
Both solutions were allowed to stir for an additional 10 minutes post the addition of TPP. It was observed that the chitosan solution went from clear to opalescent, indicating the presence of nanoparticles.
2 mL of each solution was analyzed at the Manipal Institute of Applied Physics, using a Particle Size Analyzer (Horiba Scientific Nano Partica SZ-100).

Results:

0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 141.7 nm
PI: 0.428
Zeta Potential (mean): 9.9 mV

0.4% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 26 mV

Conclusions:

The hydrodynamic radius of the 0.1% nanoparticle solution was ideal, but the zeta potential was significantly low. The 0.4% solution’s particle size was much higher than expected, due to the formation of aggregates and insufficient filtration. Its Zeta potential was also much lower than expected.

The Zeta potential could be increased by altering the amount of acetic acid used, whereas the stirring duration would optimise the nanoparticle size, along with proper filtration of the chitosan solution.

Fig 8. Chitosan solutions before and after the addition of TPP (left to right)(1) — Fig 8. Chitosan solutions before and after the addition of TPP (left to right)

Fig 8. Chitosan solutions before and after the addition of TPP (left to right)(2) — Fig 8. Chitosan solutions before and after the addition of TPP (left to right)

Fig 18. Experimental setup for ionic gelation — Fig 9. Experimental setup for ionic gelation

P. capsici Growth Curve 1 (2^nd August - 8^th August)

+

Colony Diameter Measurement (Growth Curve)

2^nd August (Day 7)

Growth Curve PDA Day 7 (168 hours)

Y axis: 8.3 cm
X axis: 8.3 cm

Final reading: 7.5 cm

Growth Curve Carrot Agar Day 7 (168 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

3^rd August (Day 8)

Growth Curve PDA Day 8 (192 hours)

Y axis: 8.3 cm
X axis: 8.3 cm

Final reading: 7.5 cm

Growth Curve Carrot Agar Day 8 (192 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

4^th August (Day 9)

Growth Curve PDA Day 9 (216 hours)

Y axis: 8.3 cm
X axis: 8.3 cm

Final reading: 7.5 cm

Growth Curve Carrot Agar Day 9 (216 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

5^th August (Day 10)

Growth Curve PDA Day 10 (240 hours)

Y axis: 8.3 cm
X axis: 8.3 cm

Final reading: 8.2 cm

Growth Curve Carrot Agar Day 10 (240 hours)

Y axis: 8.2 cm
X axis: 8.2 cm

Final reading: 7.4 cm

Conclusions:

Over the course of the ten days, it was observed that the P. capsici showed faster growth on the carrot agar plate as compared to the potato dextrose agar plate. The carrot agar plate showed uniform growth and was identical to the original P. capsici plate we had received from IISR, Kozhikode.

Chitosan Nanoparticle Production 3

+

Chitosan Nanoparticle Production

3^rd August

30 mL of 0.4% chitosan solution was prepared in 1% acetic acid solution. The solution was subjected to overnight stirring.

4^th August

The 0.4% chitosan solution kept for stirring overnight was opalescent, with fine suspended particles observed. This was due to improper cleaning of the beakers, which might have contained residual TPP solution.
A fresh 0.4% chitosan solution was prepared and kept for overnight stirring.
As seen in Fig 10., the chitosan solution became foamy and opaque after stirring overnight.

Fig 10. Chitosan solution after magnetic stirring overnight

Chitosan Nanoparticle Production 4

+

Chitosan Nanoparticle Production

5^th August

The overnight 0.4% chitosan solution was observed to be foamy the next morning, and therefore passed through filter paper.
TPP solution was freshly prepared and passed through a 0.22 μm PVDF filter.
6 mL of TPP solution was added to 20 mL of the 0.4% chitosan solution using a burette. On average, the time between drops was adjusted to 7–10 seconds.
The solution was allowed to stir throughout the process, and for an additional 25 minutes post the addition of TPP.
The samples were tested using the Particle Size Analyzer.

Results:

0.4% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 1 mV

Subsequently, two chitosan solutions (0.1% chitosan in 1% acetic acid and 0.2% chitosan in 1% acetic acid) were prepared to further optimize the concentrations for nanoparticle production.

Conclusions:

The 0.4% chitosan solution was ruled out for nanoparticle production, as aggregation was inferred from the Particle Size Analyzer results.

Chitosan Nanoparticle Production 5

+

Chitosan Nanoparticle Production

7^th August

The overnight 0.1% chitosan solutions were filtered.
A 2.4 mg/mL TPP solution was prepared and filtered using a 0.22 μm PVDF filter.
A glass column with precise flow control was used to maintain a constant flow rate of 7.2 seconds between TPP drops. The 1:2 and 1:3 TPP to chitosan solutions were prepared by adding 7.5 mL and 5 mL of TPP to 15 mL of chitosan solutions, respectively.

Fig 11. Glass column setup to achieve the ideal TPP flow rate
Both solutions were subjected to magnetic stirring throughout the process. They were allowed to stir for an additional 20 minutes post-TPP addition.
The samples were tested using the Particle Size Analyzer.

Results:

0.1% Chitosan Solution in 1% Acetic Acid (1:2 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 16.3 mV

0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 1808.0 nm
PI: 2.515
Zeta Potential (mean): 9.8 mV

Fig 12. The chitosan nanoparticle solutions that were subjected to analysis

Subsequently, two 30 mL 0.1% chitosan solutions of 1.22% and 0.8% acetic acid were prepared. The solutions were stirred overnight.

Conclusions:

The non-ideal values for polydispersity, hydrodynamic radius, and zeta potential could be due to insufficient mixing during nanoparticle formation. A fine suspension observed in the solutions indicates the same. The solutions must be subjected to sonication after the addition of TPP.

Chitosan Nanoparticle Production 6

+

Chitosan Nanoparticle Production

8^th August

The overnight chitosan solutions were passed through filter paper.

Fig 13. Foamy chitosan solution before filtering
A 2.4 mg/mL TPP solution was prepared and filtered through a 0.22 μm PVDF filter.
The addition of TPP was done using the glass column, with a constant flow rate of 8 seconds between drops. 6 mL of TPP was added to each of the 20 mL chitosan solutions to make 1:3 nanoparticle solutions.
The solutions were stirred throughout the process. They were allowed to spin for 20 minutes post-TPP addition.
The samples were tested using the Particle Size Analyzer.

Results:

0.1% Chitosan Solution in 0.8% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 14.4 mV

0.1% Chitosan Solution in 1.22 % Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 16.5 mV

Subsequently, two chitosan solutions (0.1% chitosan in 1% acetic acid) were prepared. The volume was increased to 40 mL to account for losses during overnight stirring.

Conclusions:

A fine suspension was observed yet again in the solutions, indicating insufficient mixing during ionic gelation. This confirms that sonication of the sample for a period of 10 minutes (10 seconds ON/OFF) at 50% amplitude is necessary.

P. capsici Subculturing 2

+

P. capsici Subculturing 2

8^th August

The LAF and apparatus (inoculation loops, carrot agar plates, cork borer, etc) were cleaned and sterilized through UV treatment.
Mycelial discs from the primary culture were made and inverted onto 3 fresh carrot agar plates, using the cork borer and inoculation loops. These plates were used to study the growth of the culture.
One plate was set with 2 mycelial discs to serve as the subculture.
The plates were then placed in the dark incubator at 28°C.

Chitosan Nanoparticle Production 7

+

Chitosan Nanoparticle Production

9^th August

Chitosan nanoparticles were prepared using 0.1% and 0.2% chitosan solutions, keeping the acetic acid concentration constant, and a chitosan to TPP ratio of 1:3.
The overnight stirred chitosan solutions of 0.1% and 0.2% were used. The 0.1% solution was less foamy.
2.4 mg/mL of TPP solution was prepared. The solution was filtered using a 0.22 μm PVDF filter. A total volume of 40 mL of chitosan in acetic acid was subjected to magnetic stirring, and 30 mL was measured to make the nanoparticles and account for any losses during filtration.
Using the glass cylinder with a valve, a constant flow rate of 8 seconds was maintained between consecutive drops. Both solutions were stirred using separate magnetic stirrers, and after adding TPP, they were allowed to spin for an additional 20 minutes.
The samples were then sonicated for 10 minutes with 50% amplitude and a 10-second ON/OFF pulse cycle.

Results:

0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 146.5 nm
PI: 0.287
Zeta Potential (mean): 13.5 mV

0.2% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 21.1 mV

0.1% Chitosan solution in 0.8% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 148.1 nm
PI: 0.471
Zeta Potential (mean): 15.1 mV

0.1% Chitosan solution in 1.22% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 113.3 nm
PI: 0.376
Zeta Potential (mean): 19.7 mV

Conclusions:

A fine suspension of particles was observed in both solutions, indicating insufficient mixing.

The chitosan solutions must be subjected to sonication for 10 minutes, with 10 seconds ON/OFF off pulse at 60% amplitude.

The nanoparticle samples made on 8^th August were also subjected to sonication with the same parameters before characterization.

P. capsici Growth Curve 2

+

Colony Diameter Measurement (Growth Curve)

9^th August (Day 1)

Growth Curve Carrot Agar Plate 1 (24 hours)

Y axis: 1.7 cm
X axis: 2.05 cm
Final reading: 1.875 cm

Growth Curve Carrot Agar Plate 2 (24 hours)

Y axis: 2.35 cm
X axis: 2.45 cm
Final reading: 2.4 cm

Growth Curve Carrot Agar Plate 3 (24 hours)

Y axis: 2.55 cm
X axis: 2.4 cm
Final reading: 2.475 cm

10^th August (Day 2)

Growth Curve Carrot Agar Plate 1 (48 hours)

Y axis: 4.4 cm
X axis: 4.9 cm
Final reading: 3.85 cm

Growth Curve Carrot Agar Plate 2 (48 hours)

Y axis: 5.4 cm
X axis: 4.8 cm
Final reading: 4.3 cm

Growth Curve Carrot Agar Plate 3 (48 hours)

Y axis: 5.2 cm
X axis: 4.5 cm
Final reading: 4.05 cm

11^th August (Day 3)

Growth Curve Carrot Agar Plate 1 (72 hours)

Y axis: 7.4 cm
X axis: 7.35 cm
Final reading: 6.575 cm

Growth Curve Carrot Agar Plate 2 (72 hours)

Y axis: 7.8 cm
X axis: 7.4 cm
Final reading: 6.8 cm

Growth Curve Carrot Agar Plate 3 (72 hours)

Plate 3 was observed to contain visible contamination. The plate was decontaminated and discarded.

Detached Leaf Assay 1

+

Detached Leaf Assay

12^th August

Three plastic, transparent boxes were bought from the store and were wiped thoroughly with 70% ethanol.
Only the bottom of the boxes were lined with moistened cotton to ensure that the sides were still see-through.
Piper nigrum Paniyur 1 variety leaves were taken from the campus. The third leaf was taken precisely from the growing tip to ensure uniform maturity. These leaves were cleaned with 70% ethanol and placed flat on the moistened cotton.
The subcultures used in all cases were 68 hours old and were grown in carrot agar plates.
There were six leaves plucked, and the assay was performed in duplicates. One was for the control, where only the leaf and the oomycete were placed. The second one was to test the effect of 1% acetic acid alone.
The third was to test the 0.1% chitosan in 1% acetic acid solution (1:3 TPP to CS) nanoparticles.
A sterile one-time use needle was heat-flamed and used to poke 10-20 holes across the centre of the leaf. The needle was then discarded.
A 5 mm mycelial disk from subculture was placed at the centre so that the mat made contact with the leaf surface. Two leaves were inoculated in this manner, serving as the control.
For the second set, 1 mL of 1% acetic acid solution was sprayed on two leaves using a spray bottle, which was sterilized earlier. Then the same inoculum was made.
For the third set, 1 mL of 0.1% chitosan in 1% acetic acid with 1:3 TPP to CS nanoparticle solution was sprayed on two leaves. The leaf was then inoculated.
The boxes were sealed tightly and placed in the sunlight. This is called the Cabin Sequester Method. The lesions were then observed and measured periodically.

13^th August

Observation:

The lesions on the detached leaf assay performed yesterday were observed. The boxes had moisture in them as there was visible mist on the inner surfaces.

There was a black spot underneath the plug on the control leaves, specifically visible in the right one.

Visible mycelial growth was observed on the leaf treated with acetic acid.

The leaf treated with chitosan nanoparticles (0.1% CS in 1% AA with 1:3 TPP to CS) remained the same compared to the time of inoculation.

These zoospore induction petri dishes were observed, and the plugs had absorbed the water.

15^th August (48 hrs)

Control leaf (left) -

X axis 1.8 cm
Y axis 1.4 cm

Control leaf (right) -

X axis 0.9 cm
Y axis 1.3 cm

The leaves sprayed with 1% acetic acid and 0.1% chitosan solution showed no lesion formation. However, mycelial growth was observed on the leaves sprayed with acetic acid.

Fig 14. Control leaves(1) — Fig 14. Control leaves

Fig 14. Control leaves(2) — Fig 14. Control leaves

Fig 15. Leaves sprayed with 1% Acetic acid

Fig 16. Leaves sprayed with 0.1% chitosan nanoparticle solution

P. capsici Sporulation 1

+

Zoospore Induction

12^th August

Two autoclaved petri dishes were filled halfway with autoclaved distilled water and kept with the lid facing upward.
Ten-fifteen 5 mm mycelial disks were cut on a subculture plate approximately 68 hrs old.
These plugs were suspended in the petri dish with water so that the mat faced upwards and the agar chunk remained soaked in the water.
The plates were parafilmed carefully and placed for incubation with illumination at 26°C.

Fig 17. Zoospore induction - Mycelial plugs kept for illuminated incubation.

14^th August

The previously made 0.1% chitosan in 1% acetic acid solution (1:3 TPP to chitosan ratio) was filtered.

The two sporulation plates kept for 48 hrs of light incubation were given a cold shock treatment by keeping them in the 4°C fridge for 30 minutes.

The zoospore solution from these plates was used to make six Eppendorf vials:

Two tubes contained the zoospore solution (1 mL pipetted out, which was later aliquoted to 100 μL)
Two tubes contained zoospore solution + 1% acetic acid solution (1 mL zoospore solution + 0.5 mL acetic acid)
Two tubes contained zoospore solution + the 0.1% chitosan nanoparticle solution (1 mL zoospore solution + 0.5 mL chitosan NP solution)

100 μL of Methylene blue dye was added to the vials containing only the 100 μL zoospore solution & the solutions were observed using a haemocytometer.

Fig 18. Observation of the zoospore solution under a microscope

Conclusions:

The amount of methylene blue added was too much to visualize the staining properly. We were advised to add a drop or two to the 100 μL of zoospore solution.

Chitosan Nanoparticle Production 8

+

Chitosan Nanoparticle Preparation

12^th August

Re-sonication of CSNP samples from 9^th and 10^th August and their characterization.

The CSNP samples prepared earlier, which were sonicated once already at 50% amplitude with a 10-second ON/OFF pulse for 20 minutes, were to be subjected to the same process again.
The parameters were tweaked this time to be 60% amplitude, 10 seconds ON/OFF for 20 minutes. These were sent for characterization using a Particle Size Analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 146.5 nm
PI: 0.287
Zeta Potential (mean): 12.2 mV

0.2% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 21.1 mV

0.1% Chitosan solution in 0.8 % Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 148.1 nm
PI: 0.471
Zeta Potential (mean): 15.1 mV

0.1% Chitosan solution in 1.22 % Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 113.3 nm
PI: 0.376
Zeta Potential (mean): 19.7 mV

Conclusions:

Though the hydrodynamic radius seemed to be around the expected range, the Zeta potential values weren’t as anticipated, even after sonication. It was then established that the pH should be measured after every step to gauge the stability.

Chitosan Nanoparticle Production 9

+

Chitosan Nanoparticle Production

12^th August

Two solutions of 30 mL of 0.1% chitosan in 1% acetic acid were made. They were magnetically stirred for 30 minutes.
Following this, they were sonicated at 60% amplitude with a 10-second ON/OFF pulse for 20 minutes.
These were then stored at 4°C.

Detached Leaf Assay 1

+

Detached Leaf Assay

16^th August (72 hrs)

Control leaf (left) -

X axis: 1.9 cm
y axis: 2.15 cm

Control leaf (right) -

X axis: 0.9 cm
Y axis: 1.5 cm

Fig 19. Control leaves (1) — Fig 19. Control leaves

Fig 19. Control leaves (2) — Fig 19. Control leaves

Fig 20. Leaves sprayed with 1% Acetic acid

Fig 21. Leaves sprayed with 0.1% chitosan nanoparticle solution

17^th August (96 hrs)

Control leaf (left) -

X axis: 2.3 cm
Y axis: 2.95 cm

Control leaf (right) -

X axis: 1.1 cm
Y axis: 1.5 cm

Fig 22. Control leaves (1) — Fig 22. Control leaves

Fig 22. Control leaves (2) — Fig 22. Control leaves

Fig 23. Leaves sprayed with 1% Acetic acid

Fig 24. Leaves sprayed with 0.1% chitosan nanoparticle solution

Chitosan Nanoparticle Production 9

+

Chitosan Nanoparticle Production

19^th August

Two solutions of 0.1% chitosan in 1% acetic acid were prepared earlier and kept. The TPP solution was also prepared beforehand.
The chitosan solutions were sonicated for 10 minutes with a 10 seconds ON/OFF pulse at 60% amplitude.
The chitosan solution's pH was measured after the same. It was found to be 2.71. While loading one of the samples into the pocket pH meter, the pipette was contaminated with an unknown liquid, which was also accidentally introduced into the sample. Due to this unfortunate event, we discarded one of the two chitosan solutions. Following this, the viable sample was filtered.
The pH of TPP was also measured, and it turned out to be 8.53. Post which the TPP was syringe filtered.
The burette was rinsed with TPP, and 10 mL was filled. A beaker with 30 mL chitosan solution (1:3 TPP to chitosan ratio) was spun at the maximum stable flow rate using a magnetic stirrer. TPP was added to the periphery at the highest possible flow rate.
The solution was allowed to stir for the next 20 minutes. This was then sonicated with the same parameters as earlier.
The samples were then sent for characterization using a Particle Size Analyzer.

Results:

0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 1.9 mV

Conclusions:

It has been established that the ideal flow rate for TPP addition is 6-8 seconds, and the magnetic stirring has to be on the maximum possible limit with the highest vortex effect, ensuring no splashing.

The reason for the unsuccessful nanoparticle production was the rapid flow rate at which TPP was added.

After discussing with our PI, Dr. Ritu Raval, we were advised to produce another nanoparticle in duplicates the following day (0.1% CS, 1% acetic acid, 1:3 TPP to CS).

Chitosan Nanoparticle Production 10

+

Chitosan Nanoparticle Production

20^th August

Two 0.1% chitosan solutions were made in 1% acetic acid.
The solutions underwent magnetic stirring for 30 minutes and were sonicated for 10 minutes each, with a 10-second ON/OFF pulse at 60% amplitude. The TPP solution was freshly prepared as well.
The pH of the acetic acid, TPP, and chitosan solutions were measured using a pocket pH meter and were found to be 1.51, 9.18, and 2.23, respectively.
The solutions were filtered afterwards, and 10 mL of the TPP solution was loaded onto the burette. 30 mL of the chitosan solution was placed on a magnetic stirrer below, to obtain the desired 1:3 TPP to chitosan ratio.
The TPP was added dropwise (7-second interval between drops), after which the solution was stirred for 20 minutes. One of the samples was lost at this stage due to human error.
The remaining sample was then sonicated at the same parameters and sent for characterization using a Particle Size Analyzer.

Results:

0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 10.2 mV

Conclusions:

Upon consulting our PI regarding the results, several optimizations to the protocol were suggested. The pH of the chitosan solution was to be adjusted to between 4 and 4.5, using 1N NaOH. An additional centrifugation and decanting step was suggested post-nanoparticle formation to obtain the ideal zeta potential.

Chitosan Nanoparticle Production 11

+

Chitosan Nanoparticle Production

21^st August

A 1% acetic acid solution was prepared, with a pH of 2.63. On preparing two 40 mL, 0.1% chitosan solutions, which underwent magnetic stirring (Sample 1 using Trishul magnetic stirrer, Sample 2 using Eltech magnetic stirrer) for 30 minutes, their pH was adjusted from 2.63 to 4.01. This was done by adding 2150 μL of 1 N NaOH to 35 mL of the chitosan solution.
The solutions underwent sonication (5 minutes, 10 seconds ON/OFF pulse, 40% amplitude) at optimized parameters.
The TPP and chitosan solutions were filtered and loaded onto the burette and beaker. The TPP was added to the chitosan solutions dropwise in a 1:3 ratio of TPP to chitosan solution, at the ideal flow rate of 7 seconds between drops. Following this, the nanoparticle solutions were stirred for 20 minutes.
After nanoparticle formation, the solutions underwent sonication for 5 minutes at 40% amplitude with a 10 seconds ON/OFF pulse cycle.
The solutions underwent centrifugation for 5 minutes at 1000 rpm at 26°C. The solutions were then decanted using a glass pipette while preserving the Falcon tube's orientation from the centrifuge.
These solutions were then characterized using a Particle Size Analyzer.

Results:

Sample 1- 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 267.2 nm
PI: 0.363
Zeta Potential (mean): 80.4 mV

Sample 2- 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 78.5 nm
PI: 0.237
Zeta Potential (mean): 10.3 mV

Conclusions:

Upon consulting our PI regarding the results, specific optimizations to the protocol were suggested. After adding TPP to the chitosan solution, the centrifugation must be 8 minutes rather than 5 to adjust the size. Based on the results, we were advised to use the Eltech magnetic stirrer for future nanoparticle preparations.

P. capsici Growth Curve 3

+

Colony Diameter Measurement (Growth Curve)

25^th August

Carrot agar media was prepared by combining 20 g of grated carrot and 1.8 g agar to observe the growth of P. capsici.

Potato dextrose agar was also prepared using 2.4 g of potato dextrose broth and 1.8 g of agar, to compare the growth on the two media.

Carrot agar and PDA were poured into 4 petri dishes, and were left in the 4°C fridge.

26^th August

The plates were placed in the LAF. The P. capsici culture inoculated on 8th August was used to make the mycelial plugs, with the help of an 8 mm cork borer. The plugs were placed mycelium with the mycelium facing down on the plates.

Two PDA plates were inoculated with a single plug at the centre for growth measurements. The same was done for two carrot agar plates.

Additionally, a carrot agar plate was prepared with two mycelial plugs on opposite sides. This would serve as the fresh subculture plate.

The plates were then parafilmed and incubated at 28°C.

Fig 25. Preparation of fresh media plates for the subculture and growth curve

27^th August (Day 1)

Growth Curve Carrot Agar Plate 1 Day 1 (24 hrs)

Y axis: 3.45 cm
X axis: 3.5 cm
Final reading: 2.675 cm

Growth Curve Carrot Agar Plate 2 Day 1 (24 hrs)

Y axis: 4 cm
X axis: 3.35 cm
Final Reading: 2.825 cm

Growth Curve PDA Plate 1 Day 1 (24 hrs)

Y axis: 2.15 cm
X axis: 2.1 cm
Final Reading: 1.325 cm

Growth Curve PDA Plate 2 Day 1 (24 hrs)

Y axis: 2.3 cm
X axis: 2.1 cm
Final Reading: 1.4 cm

Fig 26. Carrot agar plates 1 and 2 (24 hrs old)(1) — Fig 26. Carrot agar plates 1 and 2 (24 hrs old)

Fig 26. Carrot agar plates 1 and 2 (24 hrs old)(2) — Fig 26. Carrot agar plates 1 and 2 (24 hrs old)

Fig 27. PDA plates 1 and 2 (24 hrs old)(1) — Fig 27. PDA plates 1 and 2 (24 hrs old)

Fig 27. PDA plates 1 and 2 (24 hrs old)(2) — Fig 27. PDA plates 1 and 2 (24 hrs old)

28^th August (Day 2)

Growth Curve Carrot Agar Plate 1 Day 2 (48 hrs)

Y axis: 5 cm
X axis: 4.7 cm
Final reading: 4.05 cm

Growth Curve Carrot Agar Plate 2 Day 2 (48 hrs)

Y axis: 5.3 cm
X axis: 4.9 cm
Final Reading: 4.3 cm

Growth Curve PDA Plate 1 Day 2 (48 hrs)

Y axis: 3.2 cm
X axis: 3.3 cm
Final Reading: 2.45 cm

Growth Curve PDA Plate 2 Day 2 (48 hrs)

Y axis: 3.2 cm
X axis: 3.1 cm
Final Reading: 2.35 cm

Fig 28. Carrot agar plates 1 and 2 (48 hrs old)(1) — Fig 28. Carrot agar plates 1 and 2 (48 hrs old)

Fig 28. Carrot agar plates 1 and 2 (48 hrs old)(2) — Fig 28. Carrot agar plates 1 and 2 (48 hrs old)

Fig 29. PDA plates 1 and 2 (48 hrs old)(1) — Fig 29. PDA plates 1 and 2 (48 hrs old)

Fig 29. PDA plates 1 and 2 (48 hrs old)(2) — Fig 29. PDA plates 1 and 2 (48 hrs old)

29^th August (Day 3)

Growth Curve Carrot Agar Plate 1 Day 3 (72 hrs)

Y axis: 6.4 cm
X axis: 6.5 cm
Final reading: 5.65 cm

Growth Curve Carrot Agar Plate 2 Day 3 (72 hrs)

Y axis: 6.9 cm
X axis: 6.0 cm
Final Reading: 5.65 cm

Growth Curve PDA Plate 1 Day 3 (72 hrs)

Y axis: 4.6 cm
X axis: 4.4 cm
Final Reading: 3.7 cm

Growth Curve PDA Plate 2 Day 3 (72 hrs)

Y axis: 4.7 cm
X axis: 4.5 cm
Final Reading: 3.8 cm

Fig 30. Carrot agar plates 1 and 2 (72 hrs old)(1) — Fig 30. Carrot agar plates 1 and 2 (72 hrs old)

Fig 30. Carrot agar plates 1 and 2 (72 hrs old)(2) — Fig 30. Carrot agar plates 1 and 2 (72 hrs old)

Fig 31. PDA plates 1 and 2 (72 hrs old)(1) — Fig 31. PDA plates 1 and 2 (72 hrs old)

Fig 31. PDA plates 1 and 2 (72 hrs old)(2) — Fig 31. PDA plates 1 and 2 (72 hrs old)

Chitosan Nanoparticle Production 12

+

Chitosan Nanoparticle Production

23^rd August

Two 0.1% chitosan solutions were prepared in 40 mL of 1% acetic acid. Their pH was adjusted from 2.63 to 4.01. This was done by the addition of 2150 μL of 1 N NaOH. Prior to adding the NaOH to the chitosan solutions, an estimate of volume was acquired by directly adding NaOH to acetic acid.
The solutions underwent magnetic stirring for 30 minutes and sonication for 5 minutes at a 10 seconds ON/OFF pulse cycle at 40% amplitude.
The 2.4 mg/mL TPP and chitosan solutions were filtered and loaded onto the burette and beaker, respectively. The TPP was added dropwise after adjusting the flow rate to 7 seconds between drops, following which the nanoparticle solution was stirred for 20 minutes.
A second round of ultrasonication was performed for 5 minutes at a 10 seconds ON/OFF pulse cycle at 40% amplitude.
After this, the 2 solutions underwent centrifugation for 5 minutes at 1000 rpm at 26°C. The supernatant was collected while preserving their orientation as they would be in the centrifuge (to prevent disturbing the invisible pellet of macroparticles) using a glass pipette. These solutions were characterized using a particle size analyzer.

Fig 32. pH of the final nanoparticle solution

Results:

A. 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 68.7 nm
PI: 0.186
Zeta Potential (mean): 88.5 mV

B. 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 527.1 nm
PI: 0.316
Zeta Potential (mean): 46.3 mV

Conclusions:

The zeta potential obtained is ideal, but the size has to be adjusted. To tackle this, we shall use a single magnetic stirrer throughout to maintain uniformity. Additionally, the sonication time post-nanoparticle formation should be increased to 8 minutes.

Chitosan Nanoparticle Production 13

+

Chitosan Nanoparticle Production

28^th August

1% acetic acid solution was prepared, with the pH being 2.69. On preparing a 40 mL, 0.1% chitosan solution, which underwent magnetic stirring (using the Eltech stirrer) for 30 minutes, its pH was adjusted from 2.94 to 4.00. This was done by the addition of 1900 μL of 1 N NaOH to 35 mL of the chitosan solution.
The solution underwent sonication for 5 minutes, 10 seconds ON/OFF, 40% amplitude.
The TPP and chitosan solutions were filtered and loaded onto the burette and beaker, respectively. The TPP was added to the chitosan solution dropwise in a 1:3 ratio of TPP to chitosan solution, at the ideal flow rate of 7 seconds between drops, following which the nanoparticle solution was stirred for 20 minutes.
Post nanoparticle formation, the solution then underwent sonication for 8 minutes at 40% amplitude with a pulse of 10 seconds ON/OFF.
The solution underwent centrifugation for 5 minutes at 1000 rpm at 26°C. It was then decanted using a glass pipette while preserving the Falcon tube's orientation out of the centrifuge. This solution was then characterized using a particle size analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 21.7 mV
PI: 0.303
Z-average: 154.1 nm

Conclusions:

The zeta potential obtained is lower than ideal, but the size and PI are optimal. To tackle this, the pH of the chitosan solution must be brought slightly higher than 4, and this is to be tried again.

Chitosan Nanoparticle Production 14

+

Chitosan Nanoparticle Production

29^th August

A 1% acetic acid solution was prepared, with a pH of 2.7. On preparing a 40 mL, 0.1% chitosan solution, which underwent magnetic stirring using an Eltech magnetic stirrer for 30 minutes. The pH of the solution was adjusted from 2.87 to 4.09 by adding 1800 μL of 1 N NaOH to 35 mL chitosan solution.
The solution underwent sonication (5 minutes, 10 seconds ON/OFF, 40% amplitude) at optimized parameters.
The TPP and chitosan solutions were filtered and loaded onto the burette and beaker. The TPP was added to the chitosan solutions dropwise in a 1:3 ratio of TPP to chitosan solution, at the ideal flow rate of 7 seconds between drops, following which the nanoparticle solutions were stirred for 20 minutes.
Due to a lack of time, the sample could not be sonicated, centrifuged, and further sent for characterization.
The sample was stored at 4°C for further processing the next day.

Chitosan Nanoparticle Production 14

+

Chitosan Nanoparticle Production

30^th August

The sample made on the previous day was sonicated for 8 minutes at 40% amplitude with a pulse of 10 seconds ON/OFF.
The solution underwent centrifugation for 5 minutes at 1000 rpm at 26°C. It was then decanted using a glass pipette while preserving the Falcon tube's orientation out of the centrifuge. This solution was then characterized using a Particle Size Analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 58.0 mV
PI: 0.336
Z-average: 107.0 nm

Conclusions:

The zeta potential, PI, and Z-avg were favourable. We had to replicate the nanoparticle run using DEPC-treated RNase-free water to see if there was any effect on the nanoparticle production.

P. capsici Sporulation 2

+

Zoospore Induction

2^nd September

The zoospore plates kept in the water medium for 48 hrs were cold-shocked for 30 minutes. 1 mL of both samples was collected in sterile Eppendorf tubes. The samples were visualized under 40x, and not much was observed. A few drops of methylene blue were added to each tube, and the same was visualized. Small globule-like cells were observed to be wobbling around.

Fig 33. P. capsici burst sporangium (dark blue), zoospores (small blue dots) stained in methylene blue under 40x — Fig 33. *P. capsici* burst sporangium (dark blue), zoospores (small blue dots) stained in methylene blue under 40x

Observation:

The same samples were loaded onto a Neubauer hemocytometer and observed. The table below represents the number of zoospores present in each cell of the hemocytometer when one of the 5x5 center cells was viewed under 40X.

Table 1. Hemocytometer readings

Column/Row	(1)	(2)	(3)	(4)
(1)	5	8	9	11
(2)	9	13	8	12
(3)	12	8	9	9
(4)	9	9	9	14

Calculations:

It was calculated that there were 3.85 × 10⁷ zoospores per mL of the zoospore solution.

Detached Leaf Assay 2

+

Detached Leaf Assay

2^nd September

Observation:

The detached leaf assay lesion growth readings were taken, and there were lesions developed in the chitosan nanoparticle-treated pepper leaves as well. Troubleshooting was done, and unfortunately, we had put the leaves under UV radiation, which potentially had damaged the leaves. This led us to the realization that the leaves should only be cleaned with 70% ethanol.

Preparation of DEPC Treated Water

+

Preparation of DEPC Treated Water

3^rd September

⁠999 mL of distilled water was added to a 1 L Borosil reagent bottle.
⁠In the fume hood, using a glass pipette, 1 mL of DEPC solution (Sigma Aldrich) was carefully added to the Borosil reagent bottle. Note: Proper precautions were being taken while working with DEPC, which include using a fume hood, lab coat, gloves, mask, and protective goggles.
⁠The DEPC solution was left in a shaking incubator at 28°C until the DEPC globules were completely dissolved, which took over 2 hours.
⁠The DEPC solution was stored in a dark cupboard to be used the next morning. The glass pipette used was placed in an autoclavable bag to be autoclaved the next day.

Chitosan Nanoparticle Production 15

+

Chitosan Nanoparticle Production

4^th September

A 0.1% chitosan solution was prepared in 1% Acetic acid and underwent magnetic stirring for 30 minutes. The pH of the solution was adjusted from 2.73 to 4.01.
The solution was then sonicated for 5 minutes at 10-second ON/OFF pulse and 40% amplitude.
30 mL of the chitosan solution was filtered into a beaker, after which 10 mL of TPP was filter sterilized and added dropwise using a burette at the ideal flow rate (7s between drops). Following the addition of TPP, the solution was stirred for 20 minutes.
The nanoparticle solution was then sonicated at room temperature for 5 minutes at 1000 rpm.
The resulting solution of pH 6.09 was decanted carefully and characterized using a Particle Size Analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta potential: 169 mV
Z-Average size: 95.1 nm
PI: 0.454

Conclusions:

The size, zeta potential, and PI obtained for the sample are all satisfactory. The results obtained now have to be reproducible for another nanoparticle sample.

Detached Leaf Assay 3

+

Preparation of carrot agar plates and inoculation of P. capsici

4^th September (Carrot agar plates preparation)

20 g of carrot was blended using a mortar and pestle.
60 mL of distilled water was added to the carrot puree in a beaker. The mixture was then boiled in a microwave.
The froth was discarded, after which the mixture was filtered through a muslin cloth, resulting in a clear, slightly orange solution.
The filtrate’s volume was then made up to 100 mL in a measuring flask using distilled water.
The carrot solution was combined with 1.8 g of agar in a conical flask, which was autoclaved and poured onto petri dishes.

5^th September (Inoculation of P. capsici)

The LAF and apparatus (inoculation loops, carrot agar plates, cork borer, etc) were cleaned and sterilized through UV treatment.
⁠Mycelial discs from the previously made subculture plates were made and inverted onto 2 fresh carrot agar plates, using an 8 mm cork borer and an inoculation loop. Each plate was set with 2 mycelial discs.
The plates were then placed in the dark incubator at 28°C.

Chitosan Nanoparticle Production 16

+

Chitosan Nanoparticle Production

6^th September

A 0.1% chitosan solution was prepared in 1% acetic acid, which was stirred for 30 minutes. The pH of the solution was 2.91, which was not adjusted further (to optimize the pH of the final nanoparticles).
The solution underwent sonication for 5 minutes, 10-second ON/OFF pulse at 40% amplitude.
The chitosan and TPP solutions were filtered and loaded onto the beaker and burette, respectively. As done previously, 10 mL of TPP was added dropwise to 30 mL of chitosan solution at the ideal flow rate (7s between drops). Following this, the nanoparticle solution was stirred for 20 minutes.
This solution was then sonicated at the same parameters for 8 minutes, following which it was centrifuged at room temperature for 5 minutes at 1000 rpm.
The resulting solution was decanted carefully and characterized using a Particle Size Analyzer. The final pH of the chitosan nanoparticle solution was 5.9.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 184.5 mV
Z-Average size: 267.5 nm
PI: 0.488

Conclusions:

Although the pH of the nanoparticle solution was optimized and the zeta potential obtained was encouraging, the size of the nanoparticles was too large to use for siRNA encapsulation.

Chitosan Nanoparticle Production 17

+

Chitosan Nanoparticle Production

9^th September

The previous nanoparticle run with RNase-free water and an RNase-free water rinsed apparatus did not give us ideal results. The final pH of the nanoparticle solution was 5.9, without any pH adjustments using 1 N NaOH.
Upon discussion with our PI, we decided to add NaOH to get the chitosan solution's pH to 4.
Apart from this additional step, the same protocol was followed as done previously.

10^th September

The chitosan nanoparticle solution prepared the previous day was sonicated for two minutes at 40% amplitude with a 10-second ON/OFF pulse. The sample was thereafter centrifuged at 1000 rpm for 5 minutes at 26°C and decanted.

Results:

Z- avg: 130.1 nm
PI: 0.516
Zeta Potential: 89.0 mV

Conclusions:

The Z-avg, PI, and zeta potential were ideal. The zeta potential was lower than the last nanoparticle iteration, probably due to characterizing the nanoparticle 24 hours after its preparation.

P. capsici Sporulation 3

+

Zoospore Induction

8^th September

Two autoclaved petri dishes were filled halfway with autoclaved distilled water and kept upright.
⁠Ten 8 mm mycelial disks were cut on a 72 hours old culture plate.
The mycelial plugs were then suspended in the sterile water in the petri dish with the mycelium side facing upwards and agar side submerged.
The plates were parafilmed carefully and placed for incubation with illumination at 26°C.

P. capsici Sporulation

10^th September

⁠The two sporulation plates kept for 48 hours of light incubation were given a cold shock treatment by keeping them in 4°C for 30 minutes.
Six Eppendorf tubes were prepared for testing.
- Two tubes served as negative controls and each received 1 mL of zoospore suspension from the two plates, respectively.
- In two additional tubes containing 500 µL of zoospore suspension, 250 µL of 1% acetic acid was added.
- To the remaining two tubes, each containing 500 µL of zoospore suspension, 250 µL of 0.1% chitosan nanoparticle was added.
⁠The sample tubes, along with other necessary requirements, were taken to MCBR (Manipal Centre for Biotherapeutics Research) to be visualized using the upright trinocular phase contrast microscope.
A few drops of methylene blue was added to each sample tube. 10 μL of each sample was pipetted on clean glass slides and visualized at 40x and 100x.

Fig 35. P. capsici zoospores observed at 100x using the upright trinocular phase contrast microscope — Fig 35. *P. capsici* zoospores observed at 100x using the upright trinocular phase contrast microscope

Vid 1. Zoospores visualized at 100x without any treatment

Vid 2. Zoospores visualized at 100x with treatment of 1% acetic acid

Vid 3. Zoospores visualized at 100x with treatment of chitosan nanoparticle solution

Conclusions:

There was a slight change in the motility of the nanoparticle-treated zoospores compared to the control, whereas the acetic acid-treated sample was similar to the control. We were advised by our PI to use a Neubauer chamber haemocytometer to gauge and measure the effect on the motility of the zoospores between the control and treated samples by measuring the time it takes for a zoospore to move from one grid to the other.

Detached Leaf Assay 3

+

Detached Leaf Assay Lesion Observations

10^th September (24 hrs)

Untreated leaf left: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

Untreated leaf right: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

Control leaf left:

X axis: 1.3 cm
Y axis: 1.9 cm

Control leaf right:

X axis: 1.1 cm
Y axis: 1.7 cm

Acetic acid leaf left:

X axis: 0.8 cm
Y axis: 0.9 cm

Acetic acid leaf right:

X axis: 0.7 cm
Y axis: 0.7 cm

Chitosan nanoparticle leaf left:

X axis: 0.9 cm
Y axis: 0.7 cm

Chitosan nanoparticle leaf right:

X axis: 0.8 cm
Y axis: 0.1 cm

Fig 36. P. nigrum leaves without P. capsici- Negative control — Fig 36. *P. nigrum* leaves without *P. capsici*- Negative control

Fig 37. Untreated P. nigrum leaves- Control — Fig 37. Untreated *P. nigrum* leaves - Control

Fig 38. Leaves sprayed with 1% Acetic acid

Fig 39. Leaves sprayed with 0.1% chitosan nanoparticle solution

11^th September (48 hrs)

Untreated leaf left: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

Untreated leaf right: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

Control leaf left:

X axis: 2.3 cm
Y axis: 4.3 cm

Control leaf right:

X axis: 3.2 cm
Y axis: 5.6 cm

Acetic acid leaf left:

X axis: 1.9 cm
Y axis: 2.3 cm

Acetic acid leaf right:

X axis: 1.4 cm
Y axis: 1.5 cm

Chitosan nanoparticle leaf left:

X axis: 1.4 cm
Y axis: 1.7 cm

Chitosan nanoparticle leaf right:

X axis: 1.3 cm
Y axis: 1.6 cm

Fig 40. P. nigrum leaves without P. capsici- Negative control — Fig 40. *P. nigrum* leaves without *P. capsici* - Negative control

Fig 41. Untreated P. nigrum leaves- Control — Fig 41. Untreated *P. nigrum* leaves - Control

Fig 42. Leaves sprayed with 1% Acetic acid

Fig 43. Leaves sprayed with 0.1% chitosan nanoparticle solution

12^th September (72 hrs)

Untreated leaf left: (Contaminated)

X axis: 1.3 cm
Y axis: 0.8 cm

Untreated leaf right: (Contaminated)

X axis: 1.85 cm
Y axis: 2.5 cm

Control leaf left:

X axis: 7.9 cm
Y axis: 4.2 cm

Control leaf right:

X axis: 8.8 cm
Y axis: 6.4 cm

Acetic acid leaf left:

X axis: 4.7 cm
Y axis: 5.8 cm

Acetic acid leaf right:

X axis: 5.1 cm
Y axis: 8.6 cm

Chitosan nanoparticle leaf left:

X axis: 2.1 cm
Y axis: 3.4 cm

Chitosan nanoparticle leaf right:

X axis: 1.9 cm
Y axis: 2.8 cm

Fig 44. P. nigrum leaves without P. capsici- Negative control — Fig 44. *P. nigrum* leaves without *P. capsici* - Negative control

Fig 45. Untreated P. nigrum leaves- Control — Fig 45. Untreated *P. nigrum* leaves - Control

Fig 46. Leaves sprayed with 1% Acetic acid

Fig 47. Leaves sprayed with 0.1% chitosan nanoparticle solution

Detached Leaf Assay 3

+

Detached Leaf Assay Lesion Observations

13^th September (96 hrs)

Untreated leaf left:

X axis: 6.6 cm
Y axis: 10.5 cm

Untreated leaf right:

X axis: 3.1 cm
Y axis: 7.3 cm

Control leaf left:

X axis: 4.6 cm
Y axis: 8.4 cm

Control leaf right:

X axis: 8 cm
Y axis: 9.5 cm

Acetic acid leaf left:

X axis: 7.4 cm
Y axis: 11.2 cm

Acetic acid leaf right:

X axis: 8.5 cm
Y axis: 11.0 cm

Chitosan nanoparticle leaf left:

X axis: 3.9 cm
Y axis: 5.8 cm

Chitosan nanoparticle leaf right:

X axis: 2.9 cm
Y axis: 3.7 cm

Fig 48. P. nigrum leaves without P. capsici- Negative control — Fig 48. *P. nigrum* leaves without *P. capsici* - Negative control

Fig 49. Untreated P. nigrum leaves- Control — Fig 49. Untreated *P. nigrum* leaves - Control

Fig 50. Leaves sprayed with 1% Acetic acid

Fig 51. Leaves sprayed with 0.1% chitosan nanoparticle solution

14^th September (120 hrs)

Untreated leaf left:

X axis: 6.9 cm
Y axis: 9.1 cm

Untreated leaf right:

X axis: 8.1 cm
Y axis: 12.5 cm

Control leaf left:

X axis: 5.6 cm
Y axis: 9.2 cm

Control leaf right:

X axis: 8.0 cm (whole leaf)
Y axis: 9.5 cm (whole leaf)

Acetic acid leaf left:

X axis: 8.4 cm (whole leaf)
Y axis: 11.2 cm (whole leaf)

Acetic acid leaf right:

X axis: 8.5 cm (whole leaf)
Y axis: 11.0 cm (whole leaf )

Chitosan nanoparticle leaf left:

X axis: 5.4 cm
Y axis: 8.0 cm

Chitosan nanoparticle leaf right:

X axis: 3.7 cm
Y axis: 6.2 cm

Fig 52. P. nigrum leaves without P. capsici- Negative control — Fig 52. *P. nigrum* leaves without *P. capsici* - Negative control

Fig 53. Untreated P. nigrum leaves- Control — Fig 53. Untreated *P. nigrum* leaves - Control

Fig 54. Leaves sprayed with 1% Acetic acid

Fig 55. Leaves sprayed with 0.1% chitosan nanoparticle solution

15^th September (144 hrs)

Untreated leaf left:

X axis: 6.9 cm
Y axis: 9.1 cm

Untreated leaf right:

X axis: 10.1 cm
Y axis: 12.5 cm

Control leaf left:

X axis: 6.1 cm
Y axis: 9.2 cm

Control leaf right:

X axis: 8.0 cm
Y axis: 9.5 cm

Acetic acid leaf left:

X axis: 8.4 cm
Y axis: 11.2 cm

Acetic acid leaf right:

X axis: 8.5
Y axis: 11.0 cm

Chitosan nanoparticle leaf left:

X axis: 8.4 cm
Y axis: 11.2 cm

Chitosan nanoparticle leaf right:

X axis: 7.0 cm
Y axis: 9.2 cm

Fig 56. P. nigrum leaves without P. capsici - Negative
control — Fig 56. *P. nigrum* leaves without *P. capsici* - Negative control

Fig 57. Untreated P. nigrum leaves- Control — Fig 57. Untreated *P. nigrum* leaves - Control

Fig 58. Leaves sprayed with 1% Acetic acid

Fig 59. Leaves sprayed with 0.1% chitosan nanoparticle solution

Conclusions:

Over the course of 6 days, the lesions formed were observed. It was seen that the leaves treated with the chitosan nanoparticle solution showed the slowest lesion formation and lowest infection rates. In contrast, leaves treated with acetic acid and water had rapid lesion growth. The control leaves did get infected, but the infection pattern observed initially did not correlate with P. capsici; However, the infection eventually spread to cover the entire leaf. To avoid such contamination, extra measures were taken to ensure sterility is maintained.

Cytotoxicity Assay

+

siRNA Resuspension and Cytotoxicity Assay

22^nd September

1 mL of diethyl pyrocarbonate (DEPC) was added to 999 mL of distilled water to prepare DEPC water. This was carried out in the fume hood, wearing gloves, a mask, a lab coat, and protective gear like goggles. The solution was left in the shaker for 2-3 hours to ensure complete dissolution. This solution was then stored for the treating apparatus that would come in contact with the siRNA.

23^rd September

The DEPC solution prepared the previous day was used to soak the pipette tips and boxes, Falcon tubes, and Eppendorf vials that would be used to resuspend the siRNA. The apparatus was left to soak till bubbles appeared, indicating the effective action of the DEPC solution.

All of the apparatus was then autoclaved to ensure they were RNase and DEPC-free.

24^th September

siRNA Resuspension:

Calculations for various dilution concentrations were made beforehand for the resuspension of siRNA. We began resuspending the better candidate, Candidate 1, which was verified to have a greater silencing efficiency against P. capsici through our Dry Lab tests.
The supplier's stock was first spun down, and the rim was wiped with a tissue soaked in ethanol.
⁠1.5 mL Eppendorf tubes treated with DEPC were autoclaved, dried, and labelled.
Since the siRNA was fluorescence-tagged, all steps were carried out in the laminar airflow chamber with the lights off. Any tubes that contained siRNA were maintained on ice throughout. Nuclease-free water was used, and any apparatus that came in contact with the siRNA was DEPC-treated, including the tips and tip boxes. Utmost sterility was maintained throughout the procedure.
As per the supplier's data sheet, 57.8 μL of nuclease-free water was added to the master stock to obtain 57.8 μL of 100 μM stock. This was vortexed for 5 seconds and spun down again.
The 100 μM stock was further diluted into the following: 1 μM, 100 nM, 50 nM, and 1.44 nM.
The same was divided into aliquots as such: 5 tubes that contained 10 μL of 100 μM siRNA, four tubes that contained 20 μL of 1 μM siRNA, five tubes of 20 μL of 100 nM siRNA, two tubes of 25 μL of 50 nM siRNA, and one tube of 30 μL of 1.44 nM siRNA.
The standard C₁V₁=C₂V₂ formula was used to obtain the nuclease-free water value.
⁠Post this, 10 μL of all concentrations of siRNA were collected separately for a cytotoxicity assay to determine the effect of various concentrations of siRNA on P. nigrum leaves.
⁠Finally, all these Eppendorf tubes were sealed tightly with parafilm, wrapped with aluminium foil, labelled appropriately, and stored in -20°C.

Cytotoxicity Assay:

⁠Plastic containers were cleaned with 70% ethanol, lined up with a layer of cotton, and wetted with autoclaved distilled water so that no excess water was drained out while tilting the boxes. This was placed under UV rays for 20 minutes to eliminate any other form of contamination.
Piper nigrum Panniyur 1 variety leaves were cleaned with 70% ethanol and placed on the wet cotton layer. Pin pricks were made in a 4x4 pattern using autoclaved toothpicks.
⁠There was a negative control leaf, which was the one with no treatment, and other controls: leaves with pin pricks and no water added additionally, and leaves with pin pricks that were applied with 10 μL of water.
All concentrations, from the highest, 100 μM, to the least, 1.44 nM, were applied onto the leaves to test if there were any adverse effects for the siRNA.
Hence, the leaves were treated with 10 μL each of 100 μM, 1 μM, 100 nM, 50 nM, and 1.44 nM, and all the boxes, including the control, were labelled.
All pipette tips, tip boxes, and any apparatus used were DEPC-treated, and utmost sterility was maintained.
The boxes with the leaves, both control and siRNA-treated, were placed in a place with appropriate light.
Observations were taken every 24 hours for any necrosis on the leaf's surface.

25^th September (24 hrs)

Fig 61. Negative control: Leaf with no pinpricks, leaf with pinpricks, and leaf with pinpricks treated with water (left to right)

Fig 62. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to right)

Fig 63. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)

26^th September (48 hrs)

Fig 65. Negative control: Leaf with no pinpricks, leaf with pinpricks, and leaf with pinpricks treated with water (left to right)

Fig 66. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to right)

Fig 67. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)

Observation:

It was observed that none of the leaves showed any signs of necrosis, even with the 100 μM siRNA (our highest available concentration- master stock) treated leaf, proving our siRNA is 100% safe to P. nigrum.

Detached Leaf Assay 4

+

Detached Leaf Assay Preparation

24^th September

Carrot agar plates preparation:

⁠Four carrot agar plates were made using 20 g of grated carrot and 1.8% agar.
The media was autoclaved, and the plates were poured and labelled.

Inoculation of P. capsici:

⁠The LAF and apparatus (inoculation loops, carrot agar plates, cork borer, etc) were cleaned and sterilized through UV treatment.
⁠Mycelial discs from the previously made subculture plates were made and inverted onto 2 fresh carrot agar plates, using an 8 mm cork borer and an inoculation loop. Each plate was set with 2 mycelial discs.
⁠The plates were then placed in the dark incubator at 28°C.

5^th September (Inoculation of P. capsici)

The LAF and apparatus (inoculation loops, carrot agar plates, cork borer, etc) were cleaned and sterilized through UV treatment.
⁠Mycelial discs from the previously made subculture plates were made and inverted onto 2 fresh carrot agar plates, using an 8 mm cork borer and an inoculation loop. Each plate was set with 2 mycelial discs.
The plates were then placed in the dark incubator at 28°C.

Nanoformulation Production 1

+

Nanoformulation Production

25^th September

We realised that 1.44 nM was too low of a concentration to be effective against P. capsici.
Through further literature review, it was understood that 2.88 μM would be an effective concentration of siRNA, considering only 10% of it would be encapsulated. Our PI recommended we use 15 μL of 100 μM siRNA.
We first attempted a nanoparticle run without the addition of siRNA by reducing the volumes of the chitosan solution and TPP to see if we could achieve ideal results. This would have helped us gauge the volume of 100 μM siRNA required if the nanoparticle production were to be successful.
⁠Hence, a nanoparticle solution was made with our optimized protocol, where the final solution had 20 mL of 0.1% chitosan solution and 6.667 mL of 2.4 mg/ml TPP.
It was characterized the following day using a Particle Size Analyzer.
Preparation for the nanoformulation production for the following day was carried out simultaneously. All the apparatuses were treated with DEPC water by soaking them until bubbles appeared. This roughly took about 4 hours. The apparatuses were dried in the hot air oven and autoclaved for use the next day.

26^th September

The nanoparticles produced the previous day were sonicated for 2 minutes at 10-second ON/OFF pulse and 40% amplitude. The pH of the final solution was observed to be 4.23. This sample was characterized using a Particle Size Analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 20.6 mV
Z-Average size: 82.0 nm
PI: 0.426

Conclusions:

The size and polydispersity of the nanoparticles are favourable. However, the zeta potential was lower than required for siRNA encapsulation. As a result, the standard volumes of chitosan and TPP were to be used for the encapsulation of siRNA.

Cytotoxicity Assay

+

Cytotoxicity Assay

27^nd September (72 hrs)

Fig 69. Negative control: Leaf with no pinpricks, leaf with pinpricks, and leaf with pinpricks treated with water (left to right)

Fig 70. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to right)

Fig 71. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)

Nanoformulation Production 1

+

Nanoformulation Production

27^th September

The nanoformulation (siRNA encapsulated within chitosan nanoparticles) was produced using our optimized chitosan nanoparticle production protocol, with an additional step of adding 15 μL of 100 μM siRNA solution to 10 mL of the TPP solution.
All glassware and plasticware were treated with DEPC solution, dried, and autoclaved. All the reagents and solutions were prepared using RNase-free water. Measures were taken to make the experimental setup as RNase-free as possible.
The pH of the chitosan solution was adjusted to 4 using 1 N NaOH.
The final nanoformulation was sonicated for 8 minutes at 40% amplitude with a 10-second ON/OFF pulse, following which it was centrifuged at room temperature for 5 minutes at 1000 rpm.
The resulting solution was decanted carefully and characterized using a Particle Size Analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio) with encapsulated siRNA:

Zeta Potential: -4.0 mV
Z-Average size: 6410.3 nm
PI: 2.318

Conclusions:

The negative zeta potential indicated inefficient encapsulation of the siRNA within the chitosan nanoparticles. The polydispersity index was relatively high, possibly due to pH variation because of the DEPC-treated apparatus and RNase-free water used during preparation. The hydrodynamic radius was also observed to be very large. To tackle these issues, we were advised by our PI, Dr. Ritu Raval, to omit the adjustment of pH of the chitosan nanoparticle solution, simply rinse all glassware and plasticware with RNase-free water before use, and increase the sonication time of the nanoformulation from 8 minutes to 10 minutes.

Gel Retardation Assay 1

+

Gel Retardation Assay

28^th September

1 L of 1X TBE (Tris-Borate-EDTA) buffer was prepared from a 20X TBE buffer solution (50 mL of 20X TBE buffer was added to 950 mL of Milli-Q water). The buffer was added to the buffer tank.
35 mL of 2% Low EEO agarose gel was prepared using 1X TBE buffer. The agarose-TBE solution was microwaved until boiling was achieved, and the low EEO agarose had completely dissolved.
Once the solution had cooled slightly, 1.16 µL of SYBR Safe was added, and the gel was poured into the gel casting tray with the comb. A sterile micropipette tip was used to push away any bubbles formed on the gel surface. Once the gel had set, it was placed in the buffer tank, and the comb was carefully removed.
The samples to be loaded onto the gel were prepared in an aseptic and RNase-free environment, using RNase-free plasticware. To each sample (except the DNA ladder), 2 µL of RNA loading buffer was added. The samples were spun using a microcentrifuge and then loaded onto the gel.
The samples were loaded in the following order.
1. 10 µL of 1 kb DNA ladder
2. 10 µL of 50 nM siRNA
3. 10 µL of nanoformulation (prepared on 27^th September)
4. 10 µL of nanoformulation treated with 0.5 mg/mL RNase (2 µL of RNase was added to 10 µL of the nanoformulation, and the sample was incubated for 5 minutes at 56°C)
5. 10 µL of chitosan nanoparticle (prepared on 10^th September)
6. 10 µL of pre-decanted nanoformulation
The gel was allowed to run at 55 V for 2 hours, and checked in intervals.
The gel was visualized under a UV transilluminator.

Results:

Conclusions:

The gel run was unsuccessful. No siRNA was visualized in the free siRNA-loaded well or the nanoformulation-loaded wells. We decided to perform the experiment again, this time with a better nanoformulation.

Nanoformulation Production 2

+

Nanoformulation Production

29^th September

The nanoformulation (siRNA encapsulated within chitosan nanoparticles) was produced using our optimized chitosan nanoparticle production protocol, with an additional step of adding 15 μL of 100 μM siRNA solution to 10 mL of the TPP solution. The addition of TPP-siRNA solution and the steps following that were done in low light conditions to avoid quenching of our 6-FAM-tagged siRNA.
All glassware and plasticware were rinsed in RNase-free water and dried. All reagents and solutions were prepared using RNase-free water. Measures were taken to make the experimental setup as RNase-free as possible.
The pH of the chitosan solution was not adjusted to 4 using 1 N NaOH.
The final nanoformulation was sonicated for 10 minutes at 40% amplitude with a 10-second ON/OFF pulse, following which it was centrifuged at room temperature for 5 minutes at 1000 rpm. The pH of the final siRNA encapsulated nanoformulation solution was 3.24.
The resulting solution was decanted carefully and characterized using a Particle Size Analyzer.

Results:

0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio) with encapsulated siRNA:

Zeta Potential: 35.8 mV
Z-Average size: 401.7 nm
PI: 0.509

Conclusions:

The nanoformulation produced was close to ideal and had a good zeta potential, polydispersity index, and z-average values. We further experimented with the nanoformulation to calculate the entrapment efficiency of the siRNA within the chitosan nanoparticles.

Gel Retardation Assay 2

+

Gel Retardation Assay

29^th September

The same 1X TBE buffer as prepared the previous day was used for the gel run.
35 mL of 2% Low EEO agarose gel was prepared using 1X TBE buffer. The agarose-TBE solution was microwaved until boiling was achieved, and the low EEO agarose had completely dissolved.
Once the solution had cooled slightly, 1.16 µL of SYBR Safe was added, and the gel was poured into the gel casting tray with the comb. A sterile micropipette tip was used to push away any bubbles formed on the gel surface. Once the gel had set, it was placed in the buffer tank, and the comb was carefully removed.
The samples to be loaded onto the gel were prepared in an aseptic and RNase-free environment, using RNase-free plasticware. To each sample (except the DNA ladder), 2 µL of RNA loading buffer was added. The samples were spun using a microcentrifuge and then loaded onto the gel.
The samples were loaded in the following order.
1. 10 µL of 1 kb DNA ladder
2. 10 µL of 50 nM siRNA
3. 10 µL of nanoformulation
4. 10 µL of nanoformulation treated with 0.5 mg/mL RNase (2 µL of RNase was added to 10 µL of the nanoformulation, the sample was incubated for 5 minutes at 56°C)
5. 10 µL of chitosan nanoparticle (prepared on 10th September)
The gel was allowed to run at 55 V for 2 hours, and checked in intervals.
The gel was visualized under a UV transilluminator.

Results:

Conclusions:

The gel run was unsuccessful. No siRNA was visualized in the free siRNA-loaded well or the nanoformulation-loaded wells. Upon troubleshooting with our iGEM mentor, Sabyasachi Banerjee, we were advised to use autoclaved Milli-Q water for the preparation of the TBE buffer and use RNaseZap liberally to ensure the experimentation setup, glassware, and plasticware were RNase-free for our next run.

Entrapment Efficiency

+

Entrapment Efficiency of siRNA within Chitosan Nanoparticles

29^th September

We centrifuged the siRNA-loaded chitosan nanoparticle sample that was prepared the same day at 11,000 rpm and 4°C for 37 minutes.
The supernatant was carefully removed from the siRNA-loaded CS-TPP nanoparticles. To measure the absorbance of the sample, a nanocuvette was cleaned using 70% ethanol.
2 μL of 1% acetic acid was loaded as a blank in the nanocuvette, and absorbance was measured at 260 nm using a UV-Vis spectrophotometer.
2 μL of the supernatant was loaded into the nanocuvette, and absorbance was measured at 260 nm using the UV-Vis spectrophotometer (we use the Eppendorf BioSpectrometer). We ensured an RNase-free environment was maintained throughout the experiment, and cleaned the nanocuvette with 70% ethanol between uses.
The entrapment efficiency was calculated using this formula:

Entrapment Efficiency (%) = × 100

C_sample (20.778 μg/mL) is the concentration of siRNA added, and C_supernatant (2.2 μg/mL) is the siRNA concentration in the supernatant.

Fig 75. Absorbance readings of the nanoformulation

Results:

The entrapment efficiency was found to be 89.41%, indicating a high percentage of siRNA encapsulated within the chitosan nanoparticles.

Detached Leaf Assay 4

+

Detached Leaf Assay

27^th September

⁠The LAF and apparatus were cleaned and sterilized through UV treatment.
Mycelial discs from the previously made subculture plates were made and inverted onto fresh treated black pepper placed in plastic boxes lined with moist cotton, using a 5 mm cork borer and an inoculation loop.
Before inoculation of the leaves with mycelial discs, the leaves were treated with our nanoformulation, chitosan nanoparticles, and other controls were prepared.
The prepared black pepper leaf samples were as follows:
1. Untreated leaf
2. Untreated leaf with pinpricks
3. Leaf treated with 10 µL of water
4. Leaf treated with 10 µL of acetic acid
5. Leaf treated with 10 µL of chitosan nanoparticle solution (prepared on 10^th September)
6. Leaf treated with 10 µL of nanoformulation (prepared the same day)
7. Leaf treated with 5 µL of 100 µM siRNA
8. Leaf treated with 5 µL of 1 µM siRNA
9. Leaf treated with 10 µL of 100 µM siRNA (prophylactic treatment - 72 hrs old treated leaf from cytotoxicity assay)
10. Leaf treated with 10 µL of 1 µM siRNA (prophylactic treatment - 72 hrs old treated leaf from cytotoxicity assay)
11. Leaf treated with 10 µL of 100 nM siRNA (prophylactic treatment - 72 hrs old treated leaf from cytotoxicity assay)
The boxes were then placed in a spot with moderate sunlight at room temperature.
Measurements for lesions were taken every 24 hours.

28^th September (24 hrs)

Fig 76. Untreated leaves - unpricked and pricked

Fig 77. Leaves treated with water and acetic acid (left to right)

Fig 78. Leaves treated with chitosan nanoparticles and nanoformulation (left to right)

Fig 79. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right)

Fig 80. Prophylactic treatment- leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) — Fig 80. Prophylactic treatment - leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)

Fig 81. Prophylactic treatment- leaf treated with 100 μM siRNA

29^th September (48 hrs)

Untreated leaf (no lesions seen)

Untreated leaf with pin pricks (no lesions seen)

Leaf treated with water:

X axis: 1.9 cm
Y axis 3.1 cm

Leaf treated with acetic acid:

X axis: 1.5 cm
Y axis: 1.7 cm

Leaf treated with chitosan nanoparticle:

X axis: 1.5 cm
Y axis: 1.8 cm

Leaf treated with nanoformulation:

X axis: 1.8 cm
Y axis: 2.6 cm

Leaf treated with free sirna 100 µM:

X axis: 1.1 cm
Y axis: 1.5 cm

Leaf treated with free sirna 1 µM:

X axis: 1.6 cm
Y axis: 1.7 cm

Prophylactic treatment leaf 1 µM:

X axis: 0.0 cm
Y axis: 0.0 cm

Prophylactic treatment leaf 100 nM

X axis: 0.9 cm
Y axis: 1.1 cm

Prophylactic treatment leaf 100 µM

X axis: 0.6 cm
Y axis: 0.8 cm

Fig 82. Untreated leaves - unpricked and pricked

Fig 83. Leaves treated with water and acetic acid (left to right)

Fig 84. Leaves treated with chitosan nanoparticles and nanoformulation (left to right)

Fig 85. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right). — 85. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right)

Fig 86. Prophylactic treatment- leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) — Fig 86. Prophylactic treatment - leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)

Fig 87. Prophylactic treatment- leaf treated with 100 μM siRNA — Fig 87. Prophylactic treatment - leaf treated with 100 μM siRNA

30^th September (72 hrs)

Untreated leaf (no lesions seen)

Untreated leaf with pin pricks (no lesions seen)

Leaf treated with water:

X axis: 2.3 cm
Y axis: 3.8 cm

Leaf treated with acetic acid:

X axis: 1.7 cm
Y axis: 2.8 cm

Leaf treated with chitosan nanoparticle:

X axis: 2.2 cm
Y axis: 2.9 cm

Leaf treated with nanoformulation:

X axis: 2.9 cm
Y axis: 3.9 cm

Leaf treated with free siRNA 100 µM:

X axis: 1.1 cm
Y axis: 1.9 cm

Leaf treated with free siRNA 1 µM:

X axis: 1.8 cm
Y axis: 2.5 cm

Prophylactic treatment leaf 1 µM:

X axis: 0.0 cm
Y axis: 0.0 cm

Prophylactic treatment leaf 100 nM

X axis: 0.9 cm
Y axis: 1.1 cm

Prophylactic treatment leaf 100 µM

X axis: 1.1 cm
Y axis: 1.0 cm

Fig 88. Untreated leaves - unpricked and pricked

Fig 89. Leaves treated with water and acetic acid (left to right)

Fig 90. Leaves treated with chitosan nanoparticles and nanoformulation (left to right)

Fig 91. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right)

Fig 92. Prophylactic treatment- leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)

Fig 93. Prophylactic treatment- leaf treated with 100 μM siRNA

Gel Retardation Assay 3

+

Gel Retardation Assay

3^rd October

1 L of 1X TBE (Tris-Borate-EDTA) buffer was prepared from a 20X TBE buffer solution (50 mL of 20X TBE buffer was added to 950 mL of autoclaved Milli-Q water). The buffer was added to the buffer tank.
35 mL of 4% Low EEO agarose gel was prepared using 1X TBE buffer. The agarose-TBE solution was microwaved until boiling was achieved, and the low EEO agarose had completely dissolved.
Once the solution had cooled slightly, 1.16 µL of SYBR Safe was added, and the gel was poured into the gel casting tray with the comb. A sterile micropipette tip was used to push away any bubbles formed on the gel surface. Once the gel had set, it was placed in the buffer tank, and the comb was carefully removed.
The samples to be loaded onto the gel were prepared in an aseptic and RNase-free environment, using RNase-free plasticware. To each sample (except the DNA ladder), 2 µL of RNA loading buffer was added. The samples were spun using a microcentrifuge and then loaded onto the gel.
The samples were loaded in the following order.
1. 10 µL of 1 kb DNA ladder
2. 10 µL of 50 nM siRNA
3. 10 µL of 100 nM siRNA
4. 10 µL of nanoformulation (prepared on 29^th September)
5. 10 µL of nanoformulation treated with 0.5 mg/mL RNase (2 µL of RNase was added to 10 µL of the nanoformulation, the sample was incubated for 5 minutes at 56°C)
6. 10 µL of chitosan nanoparticle (prepared on 10^th September)
The gel was allowed to run at 55 V for 2 hours, and checked in intervals
The gel was visualized under a UV transilluminator

Results:

Conclusions:

The gel run was successful. A faint band of siRNA was observed in the 100 nM siRNA-loaded well. No siRNA was visualized in the nanoformulation and chitosan nanoparticle-loaded wells. The 50 nM siRNA loaded well showed no bands due to the low concentration of the siRNA.

SEM Analysis

+

Scanning Electron Microscopy (SEM)

30^th September

The chitosan nanoparticle (prepared on 10^th September ) and nanoformulation samples were first sonicated for 2 minutes at 40% amplitude with a 10-second ON/OFF pulse.
Glass coverslips were wiped with 70% ethanol.
10 μL of each sample was smeared on individual glass cover slips and left to dry completely.
The samples were taken to the Central Analytical and Instrumentation Facility (CAIF), Manipal, for analysis, but were not analyzed due to issues with the gold sputter coater.

3^rd October

The samples prepared on 30^th September were successfully gold sputtered and taped on glass slides using carbon tape.
The chitosan nanoparticle and nanoformulation samples were then analyzed using the scanning electron microscope (Oxford EDS(X-act) scanning electron microscope).

Results:

Fig 95. Chitosan nanoparticles visualized at 10,000x magnification

Fig 96. Chitosan nanoparticles visualized at 250,000x magnification

Fig 97. Chitosan nanoparticles visualized at 350,000x magnification

Fig 98. Nanoformulation (prepared on 27th September) visualized at 5000x magnification — Fig 98. Nanoformulation (prepared on 27^th September) visualized at 5000x magnification

Fig 99. Nanoformulation (prepared on 27th September) visualized at 200,000x magnification — Fig 99. Nanoformulation (prepared on 27^th September) visualized at 200,000x magnification

Fig 100. Nanoformulation (prepared on 27th September) visualized at 250,000x magnification — Fig 100. Nanoformulation (prepared on 27^th September) visualized at 250,000x magnification

Fig 101. Nanoformulation (prepared on 29th September) visualized at 6500x magnification — Fig 101. Nanoformulation (prepared on 29^th September) visualized at 6500x magnification

Fig 102. Nanoformulation (prepared on 29th September) visualized at 100000x magnification — Fig 102. Nanoformulation (prepared on 29^th September) visualized at 100000x magnification

Fig 103. Nanoformulation (prepared on 29th September) visualized at 150000x magnification — Fig 103. Nanoformulation (prepared on 29^th September) visualized at 150000x magnification

Fig 104. Nanoformulation (prepared on 29th September) visualized at 350000x magnification — Fig 104. Nanoformulation (prepared on 29^th September) visualized at 350000x magnification

The SEM analysis of our samples showed us the size and morphology of our chitosan nanoparticles and nanoformulations prepared. At magnifications higher than 100000x, the chitosan nanoparticles and nanoformulation appear as spherical beads in clusters.

It is worth noting that the sample solutions were not prepared the same day as the SEM analysis and that there was a three-day delay between the preparation of the samples on the glass coverslip and the SEM analysis (the samples were stored in a desiccator during this period to preserve them).

For our future SEM analysis, to obtain better imaging where the nanoparticles are not in clusters but are spaced out, we were advised by Dr. Archana Mahadev Rao, to dilute the sample and let the sample simply air dry on the glass coverslip.

P. capsici Sporulation 4

+

P. capsici Sporulation

2^nd October

⁠Two autoclaved petri dishes were filled with 15 mL of autoclaved distilled water and kept upright.
Ten 8 mm mycelial disks were cut from a P. capsici subculture plate.
⁠The mycelial plugs were then suspended in the sterile water in the petri dish with the mycelium side facing upwards and the agar side submerged.
⁠The plates were parafilmed carefully and placed for 48 hr incubation with illumination at 26°C.

4^th October

⁠The two sporulation plates kept for 48 hours of light incubation were given a cold shock treatment by keeping them at 4°C for 30 minutes. This released the zoospores into the solution.
Six RNase-free PCR tubes were prepared with samples for testing. The samples were as follows:
1. 10 µL of the zoospore solution.
2. 4 µL of 1% acetic acid was added to 6 µL of the zoospore solution.
3. 4 µL of chitosan nanoparticle solution (prepared on 10^th September) was added to 6 µL of the zoospore solution.
4. 4 µL of 7.23 µM of siRNA was added to 6 µL of the zoospore solution.
5. 4 µL of 100 µM siRNA was added to 6 µL of the zoospore solution.
6. 4 µL of the nanoformulation solution (prepared on 29^th September) was added to 6 µL of the zoospore solution.
⁠The sample tubes (incubated for 0.5 hours) and other necessary requirements were taken to MCBR (Manipal Centre for Biotherapeutics Research) to be visualized using the upright trinocular phase contrast microscope.
10 μL of each sample was pipetted on clean glass slides and visualized at 40x and 100x.

Vid 4. Zoospores visualized at 100x without any treatment

Vid 5. Zoospores visualized at 100x with treatment of 1% acetic acid

Vid 6. Zoospores visualized at 100x with treatment of chitosan nanoparticle solution

Vid 7. Zoospores visualized at 100x with treatment of 7.23 µM siRNA

Vid 8. Zoospores visualized at 100x with treatment of 100 µM siRNA

Vid 9. Zoospores visualized at 100x with treatment of nanoformulation solution

Conclusions:

Zoospore movement was observed in the untreated, 1% acetic acid, and chitosan nanoparticle solution-treated zoospore samples. For these samples, at 100x magnification, it was observed that the zoospores were motile and vigorously moving around the field.

The zoospore samples treated with 7.23 µM siRNA, 100 µM siRNA, and the nanoformulation showed total reduction in zoospore motility. At 100x magnification, throughout the sample at various fields of view, it was observed that all zoospores had lost their motility. This proves the silencing effect of the bZIP gene by the free siRNA and siRNA encapsulated within the nanoformulation.

Fluorescence Microscopy

+

Fluorescence Microscopy

4^th October

The two previously made sporulation plates, kept for 48 hours of light incubation, were given a cold shock treatment by keeping them at 4°C for 30 minutes. This enabled the release of the zoospores from the sporangia into the solution.
Zoospore samples for testing were prepared in RNase-free PCR tubes. Since the 6-FAM-tagged fluorescent siRNA would get quenched easily when exposed to light, darkness was maintained when preparing the samples. The samples were as follows:
1. 4 µL of 7.23 µM of siRNA was added to 6 µL of the zoospore solution.
2. 4 µL of 100 µM siRNA was added to 6 µL of the zoospore solution.
3. 4 µL of the nanoformulation solution (prepared on 29^th September) was added to 6 µL of the zoospore solution.
The samples were incubated in the dark for 1.5 hours at room temperature, giving the siRNA enough time to be taken up by the zoospores.
The samples were pipetted onto clean glass slides, and a cover slip was placed on without air bubbles.
The samples were then visualized using a fluorescence microscope at 10x, 40x, 60x, and 100x magnification at FITC wavelength (excitation wavelength around 490-495 nm, and 6-FAM has 498 nm excitation wavelength and 515 nm emission wavelength) of the fluorescence microscope (we used an Olympus IX 73 Fluorescent Microscope).

Fig 105. Sample treated with our nanoformulation. 6-FAM tagged siRNA visualized within P. capsici zoospores (round green structures) visualized at 100x magnification. The fluorescence of the zoospores indicated the successful release of the siRNA from the nanoformulation — Fig 105. Sample treated with our nanoformulation. 6-FAM tagged siRNA visualized within *P. capsici* zoospores (round green structures) visualized at 100x magnification. The fluorescence of the zoospores indicated the successful release of the siRNA from the nanoformulation

Conclusions:

Successful internalization of the 6-FAM tagged siRNA was visualized at 100x magnification within P. capsici zoospore, as indicated in the image captured.

Detached Leaf Assay 5

+

Detached Leaf Assay with Treated Zoospores Alongside Mycelial Plugs

27^th September

The LAF and apparatus (inoculation loops, carrot agar plates, cork borer, etc) were cleaned and sterilized through UV treatment.
⁠Mycelial discs from the previously made subculture plates were made and inverted onto 2 fresh carrot agar plates, using an 8 mm cork borer and an inoculation loop. Each plate was set with two mycelial discs.
⁠The plates were then placed in the dark incubator at 28°C.

30^th September

Two autoclaved petri dishes were filled with 15 mL of autoclaved distilled water and kept upright.
Ten 8 mm mycelial disks were cut from a P. capsici subculture plate.
⁠The mycelial plugs were then suspended in the sterile water in the petri dish with the mycelium side facing upwards and the agar side submerged.
⁠The plates were parafilmed carefully and placed for 48 hrs incubation with illumination at 26°C.

2^nd October

The two sporulation plates kept for 48 hrs of light incubation were given a cold shock treatment by keeping them at 4°C for 30 minutes. This released the zoospores into the solution.
The LAF and apparatus were cleaned and sterilized through UV treatment.
The zoospore solution was aliquoted into tubes and was treated with our siRNA, chitosan nanoparticles, and nanoformulation.
Before inoculating the leaves with the treated zoospore samples, the leaves were pricked and placed in plastic boxes lined with moist cotton. The zoospore samples were carefully pipetted on the pricked area of the leaf surface.
The prepared black pepper leaf samples were as follows:
1. Untreated leaf with just pricks.
2. Leaf treated with 10 µL of water.
3. Leaf treated with 10 µL of zoopore solution treated with acetic acid solution (2:1 ratio).
4. Two leaves treated with 10 µL of zoopore solution.
5. Two leaves treated with 10 µL of zoopore solution treated with chitosan nanoparticle solution (prepared on 10^th September in a 2:1 ratio)
6. Two leaves treated with 10 µL of zoopore solution treated with nanoformulation (prepared on 29^th September in a 2:1 ratio)
7. Two leaves treated with 10 µL of zoopore solution, treated with 5 µL of 1 µM siRNA.
Mycelial discs from the previously made subculture plates were made and inverted onto fresh treated black pepper leaves placed in plastic boxes lined with moist cotton, using a 5 mm cork borer and an inoculation loop.
Before inoculating the leaves with mycelial discs, the leaves were treated with our latest nanoformulation and chitosan nanoparticles.
The prepared black pepper leaf samples were as follows:
1. Leaf treated with 10 µL of chitosan nanoparticle solution (prepared on 10^th September)
2. Leaf treated with 10 µL of nanoformulation (prepared on 29^th September)
⁠The boxes were then placed in a spot with moderate sunlight at room temperature.
Measurements for lesions were taken every 24 hrs.

3^rd October

No lesions were observed on any of the leaves.

Fig 106. Untreated leaves -pricked — Fig 106. Untreated leaves - pricked

Fig 107. Leaves treated with water and acetic acid (left to right)

Fig 108. Leaves treated with zoospore suspension

Fig 109. Leaves treated with zoopores were treated with chitosan nanoparticles

Fig 110. Leaves treated with zoopores were treated with the nanoformulation

Fig 111. Leaves treated with zoopores treated with 1 µM siRNA — Fig 111. Leaves treated with zoopores were treated with 1 µM siRNA

Fig 112. Leaves treated with chitosan nanoparticles

Fig 113. Leaves treated with the nanoformulation solution

4^th October (48 hrs)

Untreated leaf (pricked):

No lesion seen

Just a water-treated leaf:

No lesion seen

Zoospore-treated leaf:

No lesion seen

Zoospore acetic acid-treated leaf:

No lesion seen

Zoospore Nanoparticle-treated leaf:

No lesion seen

Zoospore Nanoformulation-treated leaf:

No lesion seen

Zoospore-free siRNA-treated leaf:

No lesion seen

P. capsici plug and nanoformulation-treated leaf:

Left leaf (negligible):
X axis: 0.0 cm
Y axis: 1.2 cm

Right leaf:
X axis: 1.1 cm
Y axis: 1.3 cm

P. capsici plug and chitosan nanoparticle-treated leaf:

Left leaf:
X axis: 1.4 cm
Y axis: 1.3 cm

Right leaf:
X axis: 1.2 cm
Y axis: 1.3 cm

Fig 114. Leaves treated with water and acetic acid (left to right)

Fig 115. Leaves treated with zoospore suspension

Fig 116. Leaves treated with zoopores were treated with chitosan nanoparticles

Fig 117. Leaves treated with zoopores were treated with the nanoformulation

Fig 118. Leaves treated with zoopores treated with one µM siRNA — Fig 118. Leaves treated with zoopores were treated with one µM siRNA

Fig 119. Leaves treated with chitosan nanoparticles

Fig 120. Leaves treated with the nanoformulation solution

5^th October (72 hrs)

Untreated leaf (pricked):

No lesion seen

Just a water-treated leaf:

No lesion seen

Zoospore-treated leaf:

No lesion seen

Zoospore acetic acid-treated leaf:

No lesion seen

Zoospore Nanoparticle-treated leaf:

No lesion seen

Zoospore Nanoformulation-treated leaf:

No lesion seen

Zoospore-free siRNA-treated leaf:

No lesion seen

P. capsici plug and nanoformulation-treated leaf:

Left leaf (negligible):
X axis: 1.3 cm
Y axis: 1.4 cm

Right leaf:
X axis: 1.7 cm
Y axis: 2.1 cm

P. capsici plug and chitosan nanoparticle-treated leaf:

Left leaf:
X axis: 2.2 cm
Y axis: 2.1 cm

Right leaf:
X axis: 1.6 cm
Y axis: 2.1 cm

Fig 121. Leaves treated with water and acetic acid (left to right)

Fig 122. Leaves treated with zoospore suspension

Fig 123. Leaves treated with zoopores treated with chitosan nanoparticles — Fig 123. Leaves treated with zoopores were treated with chitosan nanoparticles

Fig 124. Leaves treated with zoopores were treated with the nanoformulation

Fig 125. Leaves treated with zoopores treated with 1 µM siRNA — Fig 125. Leaves treated with zoopores were treated with 1 µM siRNA

Fig 126. Leaves treated with chitosan nanoparticles

Fig 127. Leaves treated with the nanoformulation solution

Conclusions:

Over three days, visible necrosis (blackening of the leaves) was observed in the leaves treated with just the zoospore solution and zoospores treated with 1% acetic acid solution. The leaf treated with just water also showed visible necrosis due to contamination from the leaf with zoopores treated with 1% acetic acid solution (both leaves were placed in the same box). The leaves treated with zoospores with chitosan nanoparticle, 1 µM siRNA, and the nanoformulation showed minimal to no visible necrosis on the leaf surface, indicating a successful qualitative assessment of the chitosan nanoparticle, 1 µM siRNA, and the nanoformulation on P. capsici zoopores.

The leaves treated with the chitosan nanoparticles and the nanoformulation, upon which mycelial plugs were placed, showed lesion formation. It was observed that the leaves treated with the nanoformulation had smaller lesions compared to those treated with the chitosan nanoparticle.

Wet Lab Training

Wet Lab Basic Training Assessment

17th March

Wet Lab Basic Training

Competent cell preparation and Transformation

18th March to 21st May

SDS-PAGE

21st April

Colony PCR

22nd April

Plasmid Isolation

22nd May

References:

Week 1 - 25th July to 1st August

P. capsici Subculturing 1

P. capsici Subculturing 1

25th July

Preparation of Media Plates for Subculturing

Preparation of Carrot Agar:

Preparation of PDA:

26th July

Culturing P. capsici using Carrot Agar and PDA

P. capsici Growth Curve 1 (26th July - 1st August)

Colony Diameter Measurement (Growth Curve)

27th July (Day 1)

Growth Curve PDA Day 1 (24 hours)

Growth Curve Carrot Agar Day 1 (24 hours)

Conclusion:

28th July (Day 2)

Growth Curve PDA Day 2 (48 hours)

Growth Curve Carrot Agar Day 2 (48 hours)

29th July (Day 3)

Growth Curve PDA Day 3 (72 hours)

Growth Curve Carrot Agar Day 3 (72 hours)

30th July (Day 4)

Growth Curve PDA Day 4 (96 hours)

Growth Curve Carrot Agar Day 4 (96 hours)

31st July (Day 5)

Growth Curve PDA Day 5 (120 hours)

Growth Curve Carrot Agar Day 5 (120 hours)

1st August (Day 6)

Growth Curve PDA Day 6 (144 hours)

Growth Curve Carrot Agar Day 6 (144 hours)

Lactophenol Blue Staining

Lactophenol Blue Staining

26th July

Lactophenol blue staining:

29th July

P. capsici Water Storage

Water Storage for P. capsici

29th July

P. capsici water storage:

Testing Chitosan Solubility

Chitosan nanoparticle production

30th July

Chitosan nanoparticle production preparation

Conclusions:

Chitosan Nanoparticle Production 1

Chitosan Nanoparticle Production

31st July

1st August

Conclusions:

Week 2 - 2nd August to 8th August

Chitosan Nanoparticle Production 2

Chitosan Nanoparticle Production

2nd August

Results:

0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

0.4% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Conclusions:

P. capsici Growth Curve 1 (2nd August - 8th August)

Colony Diameter Measurement (Growth Curve)

2nd August (Day 7)

Growth Curve PDA Day 7 (168 hours)

Growth Curve Carrot Agar Day 7 (168 hours)

3rd August (Day 8)

Growth Curve PDA Day 8 (192 hours)

Growth Curve Carrot Agar Day 8 (192 hours)

4th August (Day 9)

Growth Curve PDA Day 9 (216 hours)

17^th March

18^th March to 21^st May

21^st April

22^nd April

22^nd May

Week 1 - 25^th July to 1^st August

25^th July

26^th July

P. capsici Growth Curve 1 (26^th July - 1^st August)

27^th July (Day 1)

28^th July (Day 2)

29^th July (Day 3)

30^th July (Day 4)

31^st July (Day 5)

1^st August (Day 6)

26^th July

29^th July

29^th July

30^th July

31^st July

1^st August

Week 2 - 2^nd August to 8^th August

2^nd August

P. capsici Growth Curve 1 (2^nd August - 8^th August)

2^nd August (Day 7)

3^rd August (Day 8)

4^th August (Day 9)

5^th August (Day 10)

3^rd August

4^th August

5^th August

7^th August

8^th August

8^th August

Week 3 - 9^th to 15^th August

9^th August

9^th August (Day 1)

10^th August (Day 2)

11^th August (Day 3)

12^th August

13^th August

15^th August (48 hrs)

12^th August

14^th August