Bioengineering of Neem Colloidal Nano-Emulsion Formulation With Adjuvant for Better Surface Adhesion and Long Term Activity in Insect Control

Nusrat Iqbal (  nusratsiddiqa20@gmail.com ) Institute Of Pesticides Formulation Technology Dipak Hazra Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur Aloke Purkait Department of Soil Science and Agricultural Chemistry, Palli-Siksha Bhavana (Institute of Agriculture), Visva Bharati, Sriniketan Natish Kumar Institute Of Pesticides Formulation Technology Jitendra Kumar Institute Of Pesticides Formulation Technology


Introduction
Eggplant (Solanum melongena L.) is one of the important vegetable crop in South and South-East Asia 1 with 37% of the total cultivated area, and 53.31 million tons) production (worldwide (FAOSTAT, 2016).Major insect pests of eggplant are shoot and fruit borer (Leucinodesorbonalis Lepidoptera: Pyralidae), leafhopper (Amrascabiguttulabiguttula, Hemiptera, Cicadelloidea), white y (Bemiciatabaci,Hemiptera: Aleyrodidae), aphid (Aphis gossypii, Homoptera: Aphididae), thrips (Thripstabaci,Thysanoptera: Thripidae), etc 2 .Sucking pests cause considerable loss of crop and also serve as vector for e transmission of various pathogens 3 .Therefore, these pests damage the crop directly or indirectly in terms of yield and quality 4,5 .Among the sucking pests, white ies are major destructive pests of brinjal 6,7 and responsible for 70-92% brinjal crop loss 8 .Indiscriminate use of pesticides usage leads to pest resistances and pest resurgence problems in eggplants 10 .Therefore, in view of these adverse effects caused by chemical pesticides there is a need to search for novel pest management strategies for long term activity and safeness toward users and environment 11 .Numerous botanochemical formulations have been developed with good plant defense mechanisms against pest attack 12,13 .Most commonly used botanical formulations are based on plant essential oils, plant extracts, or secondarymetabolites of plants 14,12,15 .Among botanical pesticides, neem has been the most effective botanical since last 30 years 16 .The main active ingredients in neem oil are Azadiractin A, Azadirachtin B, Salanine, Nimbin, etc. Azadirachtin has various biological properties like anti-feedant, growth inhibition, oviposition deterrent, etc with potentiality to kill various insect species 17 .Neem contains many constituents that possess varying degrees of stability and biological activity as a result very few neem formulations have been commercialized 18 .So, there is an urgent need to nd out effective adjutants to stabilize the active ingredients of most valuable botanical pesticides obtained from neem to give desired bio-e cacy for longer duration.
Hence in the present study attempts will make to enhance the stability and e cacy of neem-based Nanoemulsion.Thus, botanical adjuvant may give good stability of active ingredients in NE formulation.The stabilized neem NE formulation with botanical adjuvant in a kinetically stabilized the system and may also give good bio-e cacy against agricultural pests.

Nanoemulsion formulation process optimization
The oil-in-water nanoemulsion was formulated using suitable inert ingredients through low-energy emulsi cation approach.It is a spontaneous, simple and cost-effective process of formulating oil into nanoemulsion 18 .This method can be accomplished by the aqueous phase or oil phase titration process.The NE (O/W) was prepared using neem oil, lemongrass oil, surfactants, botanical adjuvant, anti-freezing agent and water (Fig. 7).The concentration of the oil phase and surfactants were varied during the preparation and ternary phase diagram based on three components: surfactant, water and oil were generated 19 .Atlox 4916 and Nonylphenol ethoxylates were selected for being non-ionic surfactants and hence least affected by pH and ionic strength 20 .The 1:5 ratio of the NE was found to be stable with droplet size (25-50nm) and polydispersity index (<0.301)(Fig. 1) which is indicative of the system 21 .Selection of optimal adjuvant blend is a key to stable nanoemulsion due to strong repulsive force that averts occulation and coalescence between the nanodroplets 22 .Lemongrass oil (Cymbopogon citratus) and Prosopis juli ora extracts were found to be suitable adjuvant for the formulation of stable NE since these are less affected by pH, and are considered to be nontoxic and biocompatible 23 .The stability of the NE can persist over longer period of time due to the presence of the stabilizing adjuvant that inhibits the coalescence of the nanodroplets. 24,25fect of lemongrass oil on kinetic stability Stability is one of the most important parameters in nanoemulsion system because of their small droplet size and large surface area.The small droplet size of nanoemulsions provides stability against sedimentation or creaming because of the Brownian motion and consequently the diffusion rate are higher than the sedimentation rate induced by the gravity force.A stability test of nanoemulsions was conducted by varying the storage time (0-14days) or temperatures (4-45 °C).The study was accomplished by observing the sample appearance or measuring their physicochemical properties such as zeta potential or particle size at predetermined interval time and the samples without any major changes in their appearance likes phase separation, creaming, occulation, coalescence and sedimentation was considered as a stable system.
In the present study, no phase separation, creaming or crystallization were observed in neem oil nano-emulsion with botanical adjuvant after storage for 14 days at low (0 °C) and high temperature (45 °C).The results indicate that neem nano-emulsion is kinetically stable formulation during storage for extended period of time 23 .
Generally nanoemulsion formation requires external energy to increase Gibbs free energy (ΔG) in nano droplets for extended stability.Suitable adjuvant system is also necessary for improved kinetic stability of nanoemulsion.Lemongrass oil has been previously reported as stabilizer and co-surfactant for neem oil microemulsion 20 .
Role of Prosopis juli ora on droplet size, poly dispersive index (PDI) and zeta potential Droplet size and zeta potential measurements are the general requirements to test the nano-emulsion stability 24 .The most common destabilization phenomenon like occulation, creaming, and coalescence etc. are directly related to droplet size 25,26 .The small size of nanoemulsion is desirable in achieving optimal e ciency.A complete picture of droplet size population and distribution in Neem NE formulations were obtained by analysis of data generated by dynamic-light-scattering (DLS).The average droplet size was found in the range of 25-50nm without any major changes in droplet size during storage.Nano-emulsions with smaller the droplet size will increase the Brownian motion and weaken the attractive forces in nano-emulsion systems to produce stable formulation 27 .During storage conditions, smaller droplets generally converted into larger droplet size by Ostwald ripening process which can be inhibited by selective surface active agents or adjuvant.Droplet growth rate is explained by the Lifshitz-Slezov and Wagner equation:-In neem nano-emulsion, average droplet radius (r) during storage time (t) are reduced due to low interfacial tension (ϒ) generated by surface active agents or adjuvant and stabilizers which ultimately reduced the molecular volume of neem oil (V m ) at absolute temperature (T).Occurrence of Ostwald ripening was avoided by increasing the elasticity of droplet 103 and the addition of natural adjuvant which reduced interfacial free energy forming a mechanical barrier against coalescence.Prosopis juli ora, may be acted as bio-polymeric adjuvant due to presence of cellulose ber 28,29 in this formulation.
Polydispersity values below 0.2 indicate uniformity among oil droplet sizes or monomodal distributions and therefore better stability, whereas values close to 1 indicate a heterogeneous or multimodal distribution 29 .In the present study, values were closer to 0.3 indicating mono-dispersive nature of developed nano-emulsion with good stability during storage.The selection of PDI value which is less than 0.5 is acceptable for agricultural use and is considered as a good uniformity of the droplet diameter.
Zeta potential values provide the information about the homogenous behavior of the nano-emulsion.Particles with zeta potential more positive than + 30 mV or more negative than -30 mV are usually considered to be stable, since electrical charge of droplets is strong enough to assume that repulsive forces between droplets are predominant in the nanoemulsion 60 .The surface of NE was negatively charged with an average zeta potential (−30 and −40mV) which indicates that the formulation is stable.A negative zeta potential value induces repulsive forces that are greater than the attraction forces among droplets, thus averting the coagulation and coalescence to occur in disperse emulsion.Zeta potential quanti es surface charge over the emulsion droplet indicating the physical stability of nano-formulations 30 .Zeta potential gives the electrophoresis mobility data related to the stability of NE formulation.Higher value indicates good stability of nano-formulation due to higher repulsion between the droplets to prevent coagulation and occulation during storage 31 .Droplet size, PDI and zeta potential signify clear, uniform and homogenous micelle based neem nanoemulsion formulation with long term stability.

Botanical adjuvant on acidity/alkalinity (pH)
The surface properties around the droplet determine the pH value as an indicator of nanoemulsion stability.The pH of the formulations was found in the slightly acidic range (5.9-6.5).However, the formulations become almost neutral at application concentration (0.1-1%).Previous studies have reported that high alkalinity or acidity of the nano-formulations leads to degradation of neem ingredient which in turn, reduces the bio-e cacy of the formulation 32 .In another study it was reported that pH aggregates and destabilize the nano-emulsion during storage 33 .In the present study, it was observed that botanical adjuvant stabilizes the pH to slightly acidic (5.9-6.5) to reduce degradation (1.42%) of neem during storage at low, ambient and high temperatures.

Viscosity
Neem NE without botanical adjuvant was found to be less viscous (88.7cPs) compared to Neem NE with botanical adjuvant (98.8cPs).Optimum viscosity of a formulation is required for long term storage without sedimentation, complete transfer from container to spray tank, homogeneous dispersion for spray solution and adhesion and reduction of run-off from target surfaces 34 .Optimum viscosity also reduces the rate of aggregation in nano-emulsion during storage 35 .The viscosity value may be affected by the nature of surfactants, organic phase components and oil viscosity.Pesticide nanoemulsion produces low viscosity as it is categorized as O/W type with high water loading.However, the viscosity of nanoemulsion can be altered by surfactant concentration 35 .Results showed that Neem NE with botanical adjuvant can give long term stability during storage and improved adhesion on applied surfaces for good bio-e cacy.

Surface tension
Low surface tension (23.4mNm -1 ) in neem NE (with adjuvant) provides better spreading of formulation on brinjal leaves with good bio-e cacy (91.24%) compared to neem NE (without adjuvant) (33.6 mNm -1 ).Finer droplet size allows the spreading of droplet uniformly on the plant leaf surface 36 .Lower surface tension improves wetting, spreading and penetration into the applied surface of plants 37 .Wetting is the important parameter which directly linked with the contact angle and surface tension.Retention and contact angle of leaves are measured to relate the a nity of the pesticide liquid towards the leaf surfaces.Contact angle is the quantitative measurement of surface adhesion of liquid on solid surface 38 .Contact angle measurement is linked to Young's equation; ------------------------------------------------------( The e ciency of neem nanoemulsion was enhanced by increasing the adhesion work of nanoemulsion towards the leaves.Neem nano-emulsion reduces the surface tension and contact angle due to spreading of oil droplets over leaf surface (Fig. 2).It worth noted that the contact angle of nanoemulsion decreased as the increasing P.juli ora content, showing that neem oil has low interfacial tension which effectively allowing neem oil diffusion in the plant surface 104 .Adjuvant along with water based neem nano-emulsion binds rmly over the leaf surface with grip induced by sticky adjuvant (Fig. 3).Neem nanoemulsion with P.juli ora conferred further lowering of surface tension may be due to the presence of cellulose polymer.So, the addition of botanical adjuvant improves wetting and dispersion of NE.

Effect of botanical adjuvant on active ingredient (azadirachtin) compatibility
FT-IR spectroscopy is a valuable technique for the identi cation of any functional group among various bioactive constituents 39 .FTIR spectroscopy of Neem nanoemulsion, neem oil and adjuvant was done and the obtained data were compared to identify the possible interaction of various functional groups that were involved in the synthesis (Fig. 4).Natural adjuvant P.juli ora showed broadband at 3314.96cm -1 due to hydroxyl groups of phenolic constituents, band at 1613cm -1 due to aromatic C=C and similar peaks have been previously reported by Kumara et.al.The study explained that functional groups were corresponds to avonoid compounds 40 .In view of previous studies, hydroxyl and aromatic peaks may be due to the avounoid constituents.Phenolic and alkene derivatives in P. juli ora extract may enhance the storage stability of azadirachtin 41 .
Neem nano-emulsion shows the corresponding peaks of neem oil, azadirachtin constituent corresponding peaks at 1745.72 cm -1 and at 2848.17 cm -1 without any chemical modi cation.FT-IR data results concluded that azadirachtin active constituents were stable and compatible with botanical adjuvant in nano-emulsion formulation system.

Effect of natural adjuvant on active ingredient (azadirachtin) storage stability
In water-based formulations, azadirachtin content is generally degraded by hydrolysis reactions (Fig. 6) during storage to reduce the bio-e cacy.So suitable stabilizer is required to protect the azadirachtin from undesirable degradation.Analytical chromatogram of neem NE formulation obtained by HPLC analysis revealed the degradation pattern of azadirachtin (Fig. 5).Azadirachtin showed linear detector response (Fig. 8) over the concentration range (0.05-1.0mgL -1 ) tested, with correlation coe cient linear functions > 0.999 and regression equations, y = 93322x -51.38.In the present study, P. juli ora acts as a stabilizing agent in neem NE formulation as results indicate that azadirachtin content remained stable with negligible degradation (1.42%) compared to NE formulation without P. juli ora (15.26%).This decrease might be due to the presence of glycosidic and phenolics compound in botanical adjuvant.
Half-life (t½) of azadirachtin in neem oil with and without of botanicals was observed and it ranged from 45.84 to 492.95 days (Table 1).The lowest t½ value was obtained without botanicals and increased manifolds with the addition of botanical synergist.An increase in the half-life of azadirachtin again signi es its stability due to the presence of botanical adjuvant.Azadirachtin is the secondary metabolite of neem, but high rate of degradation con nes its e cient usage in controlling insects 42 .Farmers are not bene tted from neem products due to its low stability as reported in various research ndings 15 .Some stabilizers have been used to reduce degradation of azadirachtin 45,46 .All these stabilizers are good and e cient but due to their chemical nature and limited availability are less popular in farmers.Whereas, in the present study, naturally occurring botanical adjuvant was used to enhance the stability of azadirachtin in neem oil which is quite safe for plants as well as for human health also.

In-vitro insecticidal activity of Neem nanoemulsion against white y
The results of in-vitro bioassay of the selected formulations against white y were presented in Table 2. Mortality (%) of white y was increased signi cantly (p <0.05) with the increment of dose and time of exposure.Neem 20 NE (with adjuvant) exhibited the best insecticidal activity against white y with LC 50 value of 2.05 mLL -1 .
Moreover, Neem 20 NE showed good mortality (56.1%) of target pest after 5days of treatment at 8mLL -1 (Table 2) which was quite better than crude neem oil at 1% dose (Pissinati and Ventura, 2015).Neem 20NE formulation (with adjuvant) demonstrating the strongest insecticidal activity with the lowest LC 50 value of 2.05mLL -1 whcih indicates that Prosopis juli ora extract acted as a promising bio-e cacy enhancer in neem nano emulsion.In-vivo insecticidal activity enhancement of neem NE against white y on brinjal Neem oil has been widely reported as potent bio-pesticide due to anti-feedant, repellent, and growth-inhibiting properties 47 .Neem oil comprises various azadirachtin analogs that offer various insecticidal properties against different agricultural as well as household insect pests 48 .Azadirachtin imparts chemical defense against pest attack for e cient pest management strategies without hampering non-target organisms.Neem oil in comparison to synthetic pesticides has been extensively studied for the control of white ies which showed equivalent control as synthetic pesticides 49 .
Nano-emulsion is a new formulation technology for improving the bio-e cacy of neem oil for long term pest control 50 .Biological activity of neem NE can be improved further by addition of adjuvant or other inert ingredients 51 .The effect of adjuvant on neem nano formulation was evaluated on white y population and percent reduction was compared with control replication wise to nullify the impact of different factors in the eld such as the presence of natural enemies, free movement of insects and conventionally used synthetic insecticide Cypermethrin 10% EC also (Table 3).Pre-treatment (PT) population of target pest was homogeneous before application and increased steadily over the period of observation till 14 days.Among formulations developed, Neem 20NE with adjuvant recorded signi cantly (p <0.05) low population of B. tabaci from 1 to 14 days after treatment @ 1000 mLha -1 .
In the present study, botanical adjuvant was used as possible bio-e cacy inducer against white ies of brinjal.Neem 20 NE (without adjuvant) reduced population (43.30%) which was further enhanced to 91.24% by incorporating botanical adjuvant and equivalent to Cypermethrin 10% EC (95.23%).Inclusion of adjuvant (P.juli ora) e ciently increased the biological effectiveness of neem nano-emulsion.Insecticidal activity of nhexane extract of P. Juli ora seed oil against termite (Odontotermes obesus) and cockroach (blattella germanica); antimicrobial activity of P. juli ora seed pods aqueous extract against some common pathogens 61 was reported earlier.Moreover, in presence of P. juli ora, neem oil nano-formulation showed possible synergistic activity (110.71%).) and untreated control (T 4 ) treatments affect the behaviour of percent reduction of white ies at different days interval (Table 4).From this study, it was found that these four categories of treatments differ in the way they reacted towards white y population management.
Test results indicated that Neem 20NE and Neem 20NE+adjuvant yielded signi cant differences (0.5203) on the reduction (%) of white ies.No signi cant difference accumulated for Cypermethrin (10% EC).It means that botanical adjuvant had strong in uence on percent reduction of white ies.

Materials
Neem oil (azadirachtin content: 2000mgL -1 ) purchased from Gujarat Chemicals, Gujrat, India.For botanical adjuvant, Kekar pods [(Prosopis juli ora, Kingdom: Plantae, Order: Fabales, Family: Fabaceae, Subfamily: Caesalpinioideae, Genus: Prosopis, Species:P.juli ora] collected from local forest nearest to the Institute.Two nonionic surfactants [Nonylphenol ethoxylate is nonionic in nature with high HLB (13); neither ionizes in water nor hydrolyzes in aqueous acid or alkaline solutions and Atlox 4916 is a high molecular weight polymeric emulsi er with low HLB (6) to provide excellent stability] were procured from Croda Surfactant Ltd, Mumbai, India.Lemongrass oil was obtained from Gorgia Chemicals, Delhi, India.Acetonitrile (HPLC grade) purchased from Merck, India.Azadirachtin standard (95% purity) procured from Sigma Aldrich, France.All the formulations were prepared using double distilled water.
Zeta potential was also measured by Malvern Zetasizer (Malvern, UK and samples were prepared by the method as described by Ch et.al, 2012 53 .The samples were dispersed in 10 mM aqueous sodium chloride (NaCl) solution 53 .All the measurements was performed in triplicates Acidity/alkalinity (pH) determination of Neem NEs The pH value of NEs was measured using a pH meter by immersing the electrode into 10% aqueous solution of formulation at 25 ± 1 °C after calibration of pH meter using buffer solutions viz., pH 7, 4 and 9.2 (CIPAC MT 75.3,  2000).All measurements were triplicated.

Rheology studies of Neem NEs
The dynamic viscosity measurements of Neem NEs (with and without botanical adjuvant) were conducted using Brook eld viscometer at 25 °C.The analyzed data were examined to observe the viscosity behavior.All the viscosity measurement was done in triplicates at room temperature.

Measurement of surface tension of Neem NEs
In the present study, surface tension of developed products was measured by Surface Tensiometer (Model: DST-30) by Elico Marketing Ltd in triplicates at ambient temperature and atmospheric pressure.The needle and the dispensing system were regularly rinsed with distilled water to prevent residue from prior experiments to impact measurements.

FTIR analysis for functional group characterization of Neem NEs
Fourier-transform infrared spectroscopy (FTIR) (PerkinElmer Spectrum) was used to study the chemical interactions between neem oil and neem oil Nano-emulsion with adjuvant by comparing their spectral absorptions.The transmittance was measured against the wave number between 500 and 4000 cm −1 .

Chemical characterization of active ingredient by HPLC in Neem NEs
Chemical constitutes were characterized by HPLC technique.Active ingredient was quanti ed in neem NE formulations (with and without adjuvant) as per BIS method 14299: 1995 with following operating conditions (Table 5).and commercially available formulation of Cypermethrin 10EC at 500 mLha -1 (T 3 ) in separate plots after observing the white y population above ETL (Economic Threshold Level).Three different plots were maintained as control (T 4 ) with absolutely no application of pesticides (only water was sprayed at 400 Lha -1 ).Each treatment was replicated thrice to minimize pest population data count error.Two round sprayings were executed by high volume knapsack sprayer at 14 days interval based on pest severity and resurgence possibility.
Observations of total number (adult +pupae) of white ies were recorded very carefully to avoid the ying of adults from three leaves per plant selected from top, middle and bottom per plot early in the morning.Before spray, rst count (pre-treatment, PT) of insect population was noted followed by post-treatment counts on 1, 3, 5, 7, 10, and 14 days after last application.

Statistical analysis
Statistical analysis was carried out by performing necessary transformation representing standard error of the mean (SEm), co-e cient of variation (CV), and critical difference (CD) at 5% level of signi cance using (ANOVA) with SPSS R software version 16.0 (SPSS Inc., Chicago, IL).Data of insect population recorded after each round of spray were pooled and analyzed.Pre-application data were used to work out % reduction of population as per Henderson-Tilton formula.Square root transformation was adopted for analysis of insect population data.Probit analysis was used to work out LD 50/90 .Post-hoc analyses were done by the Tukey test (Zar 2010).Signi cant differences among the extracts in each assay were recorded when 95% con dence intervals (CI) did not overlap.LD 50 values of the tested formulations and con dence limits were calculated for white y with log dosage-mortality probit regression equations.

Conclusion
Nano-emulsion formulation of neem oil with botanical adjuvant having standardized quality parameters was developed.Droplets of developed NE are in nano-range (25-50 nm) with zeta potential (-30mV).FTIR analysis assured the compatibility among the inert ingredients, botanical adjuvant with neem oil.Azadirachtin was substantially stable (1.42% degradation) in neem formulation with botanical adjuvant.Formulation produced comparable reduction of white y (91.24%) with synthetic pesticide (95.23%).Thus, neem oil with botanical adjuvant could be a good alternative to conventional pesticide formulations and may play a signi cant role in Integrated Pest Management (IPM) and organic cultivation.

Declarations
Ethical approval.
This manuscript does not contain any studies involving human participants or animals.

Figures
Figure 1 Representative          Detector linearity for Azadirachtin in HPLC Detector linearity for Azadirachtin in HPLC Detector linearity for Azadirachtin in HPLC Surface tension between solid and air SL= Surface tension between soilid and liquid LA= Surface tension between liquid and air Cos ϴ = Contact angle

Flow 1 .2 mLmin - 1 AA 1 =
stock solution (50 mgL -1 ) of Azadirachtin (analytical grade) used as an external standard and the retention time was recorded in HPLC.Neem NE formulations were diluted with HPLC grade acetonitrile (1:200v/v) and centrifuged at 8000 rpm for 5 min.Supernatant was ltered through 0.2μm Nylon 66 membrane lter paper with the help of syringe.Filtrate (25mL) of each cleaned up formulations was injected to HPLC.Azadirachtin was identi ed by comparing the retention time of formulation peaks with that of standard and quanti ed (mgL -1 ) as follows.Where, Area of sample in chromatogram A 2 = Area of standard in chromatogram V 1 = Total volume of sample in mL V 2 = Injected volume in µL C = Concentration of analytical standard in (mgL -1 ) W = Weight of the sample in g R f = Recovery factor In-vitro insecticidal activity of Neem nanoemulsion against white y In-vitro evaluation of insecticidal activity of the developed neem nano-emulsion against white y (Bemisia tabaci G.) of brinjal (Pusa Purple Long cultivar) was conducted using the modi ed leaf dip bioassay technique as described in prior research 62 .Freshly reared and mature (apterous adult) white ies were collected from polyhouse (27 ± 2°C and 70-80% relative humidity (RH)), under a diurnal day/night cycle of 16/8 h of Agricultural Research Farm of BCKV, Kalyani, Nadia, West Bengal (India).Associated species of white y was indenti ed and con rmed by Entomologists using identi cation keys and relevant literature.Experiment was conducted with ten (T 1 -T 10 ) treatments and three replications of Neem 20NE and Neem 20NE (with natural adjuvant).Entire brinjal leaves were dipped (30 secs) in emulsions of neem nano formulation with nal concentrations in distilled water and air dried at room temperature (27 ± 2° C).Treated leaves were placed over in separate containers and white ies obtained from polyhouse were released (60) and secured by small piece of cotton cloths using rubber bands under controlled laboratory conditions (27 ± 2°C and 70-80% relative humidity (RH)), under a diurnal day/night cycle of 16/8 h).Containers without formulation served as the negative control and chemical insecticide Cypermethrin (10% EC) as standard check for comparison.The mortality (%) of white ies was recorded at 24, 48 72 and 96 hrs of intervals and inhibition levels [mortality (%)] were calculated using the formula: [(x -y)/x] × 100-------------------(3) where x = white y population in the negative control, y = white y population in the presence of the tested formulation.LC 50 and LC 90 values ( DLS of Neem NE (without adjuvant) (A) Neem NE (with adjuvant) (B) showing hydrodynamic radius and Polydispersity index (PDI)

Figure 2 Variation of contact with surface tension variation Figure 2 Variation of contact angle with surface tension variation Figure 3 Figure 3 Figure 3
Figure 2

Table 1 :
Degradation dynamics of azadirachtin in neem NE formulations during storage.

Table 2 .
In vitro insecticidal activity of neem NEs against white y.

Table 3 :
Effect of botanical and synthetic formulations on white y infestation in brinjal crop *Values in parentheses are square-root transformed; PT: Pre-treatment; ± Compared to untreated (control) 14 days after application.Tukey's Honest Signi cant Difference (HSD) Test or Post-hoc analysis was performed to evaluate how Neem 20 NE (T 1 ), Neem 20NE+adjuvant(T 2 ), Cypermethrin 10% EC (T 3

Table 4 :
Tukey's Honest Signi cant Difference (HSD) Test of botanical and synthetic formulation's effects on *The mean difference is signi cant at the 0.05 level.

Table 5 :
HPLC operating conditions

Table 2
) of different formulations were calculated by Probit analysis(Finney, 1971).In-vivo insecticidal activity of neem oil nano-emulsion against white y on brinjal A eld experiment was conducted at the experimental farm of District Seed Farm (AB -Block Farm), Directorate of Research BCKV, Kalyani, West Bengal, from August to October 2019, with an aim to test the e cacy of the developed formulations against white y [(Bemisia tabaci Gennadius,1889) Kingdom:Animalia, Phylum:Arthropoda, Class:Insecta, Order:Hemiptera, Family:Aleyrodidae, Genus:Bemisia, Species:B.tabaci].A randomized block design (RBD) was used with three replications for each treatment and plot (5x4 m 2 ) were separated by ridges.The brinjal variety, Pusa Purple Long, was transplanted with recommended package of practices.Developed formulations were applied at 1000 mLha -1 of neem 20NE (T 1 ) neem 20NE+adjuvant (T 2 )