Helicoverpa Armigera Nuclear Polyhedrosis Virus Research Articles

Field Assessment of Insecticides as Well as Bio-Pesticides to Manage the Tomato Fruit Borer Helicoverpa armigera Hubner

A field experiment was conducted for the management of tomato fruit borer in the winter period of 2021-22 at the Vegetable Research Station of C.S.A. University, Kanpur. The current study assessed the effectiveness of some novel insecticides as well as bio-pesticides on the Azad T-6 cultivar of tomato crop. Our team used chemical insecticides such as Flubendiamide 39.35% S.C. (Suspension concentrate), Chlorantraniliprole 18.5% S.C. and Fipronil 5% S.C. and bio-pesticides viz. Spinosad 45% S.C., Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) 2% A.S. (Aqueous suspension), Bacillus thuringiensis subsp. kurstaki (Bt) 5% W.P. (Wettable powder) and Neemarin 1500 ppm (parts per million) at their suggested quantity. The observations on the number of larvae were recorded from ten arbitrarily tagged plants in each plot treated with insecticides and bio-pesticides. It was recorded that Flubendiamide 39.35% S.C. @ 0.2 ml/lit was found to be the most efficient against the fruit borer larvae trailed by Chlorantraniliprole 18.50% S.C. @ 0.3 ml/lit in decreasing the larval population and displayed maximum percent decrease in fruit infestation over untreated control. Amongst the bio-pesticides, Spinosad 45% S.C. @ 0.2 ml/lit was found to be the maximum effective over untreated control. All the used insecticides and bio-pesticides in the existing field experiment were found to be superior to the control.

(Invited) Sensors for Agricultural Pests Management

Pests are undesired living organisms which cause major threat to plants, animals, humans and other organisms. It can directly feed on the crops and indirectly spread diseases and decrease the productivity of plants. Traditionally we have been using pesticides to get rid of them, but these pesticides are chemicals which tend to accumulate in the soil and water and pose detrimental effects to all the living creatures. To reduce the major loss in food production and use of chemical pesticides, we have come up with a few innovative approaches to control pests and also detect the toxic pesticides in an easy, affordable and eco-friendly way using sensors. We had developed sensors for effective use of biopesticides such as Helicoverpa armigera nuclear polyhedrosis virus (HaNPV), Spodoptera litura nuclear polyhedrosis virus (SlNPV) and Spilosoma obliqua nuclear polyhedrosis virus (SpilNPV). We had also designed a silicon dioxide-based microelectro-mechanical system (MEMS) sensors as tool for an early selective pest detection of female sex pheromones of Helicoverpa armigera and Bactrocera oleae. This robust device can also be used to selectively measure the pheromone at femtogram levels, and it is handy for early detection of pests. The device works well in both rainy and peak summer seasons. Using these systems farmers can take necessary measures in the pest attack region without using pesticides and would be able to support organic farming.

Switchable Luminescent Probe for Trace-Level Detection of the Spodoptera litura Nuclear Polyhedrosis Virus via a Color-Changing Response

A luminescent ruthenium(II) polypyridyl complex has been designed for ratiometric optical sensing of Spodoptera litura nuclear polyhedrosis virus (SLNPV) both in aqueous medium and in commercial samples. The detailed spectroscopic studies indicate that the ruthenium(II) complex can electrostatically bind to the negatively charged outer protein (polyhedron) layer of SLNPV and renders a change in the luminescence color from red (λem ∼ 605 nm) to blue (λem ∼ 487 nm). The linear variation in luminescence color (ratiometric response) over a wide concentration range of occlusion bodies (OBs) (0–1.3 × 107 POBs/mL) ensures the quantitative estimation of SLNPV even in unknown samples. The detection limit for SLNPV was found to be 5.6 × 103 POBs/mL. In addition to discriminating the freshly prepared/active SLNPV from its storage old/less reactive analogue, the system presented here can distinguish SLNPV from other commercially available NPV pesticides, such as Helicoverpa armigera nuclear polyhedrosis virus (HaNPV). In addition, the dye-coated paper strips were developed for rapid, on-field estimation of SLNPV in commercial formulations and aqueous extracts of agricultural crops. This method can be successfully utilized to detect SLNPV in complex biological media such as leaf extracts (more than 20 different agricultural crops were screened). This sustainable strategy of detecting biopesticides is one of a kind and will surely add a new dimension to the improved management of S. litura in agriculture to minimize the extent of crop loss.

The Effect of Nanozeolite Concentration in a Delivery System of HaNPV1 to the Lethal Time against Crocidolomia pavonana

The constraints on the effectiveness of nuclear polyhedrosis virus (NPV) as biocontrol are usually due to environmental factors such as temperature and ultraviolet (UV) exposure. Zeolite has been commonly used as a carrier or delivery system for nuclear polyhedrosis viruses. In this study, zeolite powder was reduced into nanosized particles by beads milling method and was investigated for the effect of its concentration in the delivery system of Helicoverpa armigera nuclear polyhedrosis virus (HaNPV1) on the lethal time against the larvae Crocidolomia pavonana. The formulation used three concentrations of nanozeolite suspension, 0.5, 1, 1.5, and 2 wt.% applied for each 4 × 107 of HaNPV1. A randomized block design (RBD) method was applied with 3 replications. The results showed that the scanning electron microscope (SEM) from nanozeolite was seen coating the entire surface of the HaNPV1 polyhedra and an increase of zeolite concentration caused acceleration of the lethal time of C. pavonana instar III. Thus, the fastest lethal time was 1.2 days receiving a concentration of 2 wt.%, which was significantly higher compared to without delivery (2.9 days). The increase of the zeolite concentration up to 2 wt.% in the delivery system for HaNPV1 improved their performance on lethal time and mortality against C. pavonana. It was concluded that nanozeolite as a delivery system enhanced and created a synergy in infecting C. pavonana.

Safety evaluation of microbial pesticide (HaNPV) based on PCR method

Microbial pesticides can prevent and control diseases and pests of crops, and has become one of the important measures to ensure food and environmental safety. However, the potential harm of microbial pesticides to humans and animals is a serious concern at home and abroad. In this paper, we have investigated the infectivity and pathogenicity of a representative of viral microbial pesticides, helicoverpa armigera nuclear polyhedrosis virus (HaNPV), by specific and highly sensitive polymerase chain reaction technology. The results show that HaNPV can be gradually cleared in a short time after getting into blood of experimental rats, and does not infect other tissues or organs of animals; also indicate that the test subjects are not infectious to experimental rats after intravenous injection of HaNPV. Our method has good specificity and repeatability, and could provide an important reference for establishment of infectivity and pathogenicity detection methods for viral microbial pesticides in future.

Field Efficiency of Certain Biopesticides and Neem Products Against Helicoverpa armigera (Hubner) on Chickpea (Cicer arietinumL.)

A field study was conducted to evaluate the efficiency of four microbial insecticides viz. Beauveria bassiana; HaNPV (Helicoverpa armigera Nuclear Polyhedrosis virus); (Bacillus thuringiensisvar.kurstaki 2 gm/L); HaNPV+Bt; neem oil; neem cake and D.D.V.P EC 76% @0.05% at Research Farm SHIATS,Allahabad during rabi season of 2011-2012. The experiment was laid out in randomized block design with seven treatment and replicated thrice. The observation larval populations of H. armigera were recorded one day before treatment was recorded at 3,7, and 10 days after treatments. The larva population of H. armigera appeared in the third week of February (8 the Standard week) and reached its peak of 14.65 larvae in first week of April and decline rapidly with maturation of crop. There was only one peak in the larval population observation in the 1st week. Bacillus thuringiensis was the most effective chemical by D.D.V.P.76%@0.05% . Among the microbial insecticides. HaNPV ,was the most effective followed by HaNPV+Bt and neem cake . The combination treatments were less effective than the individual treatment neem oil and B. bassiana were the least effective treatment in reducing the larval population of Heliverpa armigera.

On-Field Detection of Helicoverpa armigera Nuclear Polyhedrosis Virus Using Luminescent Amphiphilic Probe: Screening of Agricultural Crops and Commercial Formulations

An easy to synthesize luminescent, amphiphilic probe has been designed for the first time in optical sensing of a biopesticide, Helicoverpa armigera nuclear polyhedrosis virus (HaNPV). The compound...

Genome-Wide Characterization of Endogenous Retroviruses in Bombyx mori Reveals the Relatives and Activity of env Genes

Endogenous retroviruses (ERVs) are retroviral sequences that remain fixed in the host genome, where they could play an important role. Some ERVs have been identified in insects and proven to have infectious properties. However, no information is available regarding Bombyx mori ERVs (BmERVs) to date. Here, we systematically identified 256 potential BmERVs in the silkworm genome via a whole-genome approach. BmERVs were relatively evenly distributed across each of the chromosomes and accounted for about 25% of the silkworm genome. All BmERVs were classified as young ERVs, with insertion times estimated to be less than 10 million years. Seven BmERVs possessing the env genes were identified. With the exception of the Orf133 Helicoverpa armigera nuclear polyhedrosis virus, the env sequences of BmERVs were distantly related to genes encoding F (Fa and Fb) and GP64 proteins from Group I and Group II NPVs. In addition, only the amino acid sequence of the BmERV-21 envelope protein shared a similar putative furin-like cleavage site and fusion peptide with Group II baculoviruses. All of the env genes in the seven BmERVs were verified to exist in the genome and be expressed in the midgut and fat bodies, which suggest that BmERVs might play an important role in the host biology.

Examination of Temperature Fluctuations on The Viability of Egyptian Isolate of Helicoverpa armigera Nuclear Polyhedrosis Virus

Examination of Temperature Fluctuations on The Viability of Egyptian Isolate of Helicoverpa armigera Nuclear Polyhedrosis Virus

Laboratory evaluation of certain bio-pesticides against the larvae of Helicoverpa armigera Hubner

Applications of Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV), Quinalphos, Bacillus thuringiensis kurstaki (Btk) and Beauveria bassiana (B.b.) as full doses gave hundred percent larval mortality to the test insect, 2nd instar larvae of Helicoverpa armigera. Treatment combinations containing half doses of HaNPV + Btk, HaNPV + B.b., HaNPV + Quinalphos, Btk + B.b., Btk + Quinalphos, B.b.+ Quinalphos, also gave cent percent larval death. Azadirachtin as half dose of application was found to be least effective (50%). However, all the treatment combinations were found effective over the untreated control.

Storage Stability and Performance of Aqueous and Dry Formulations of <I>Helicoverpa armigera</I> Nuclear Polyhedrosis Virus

Experiment on storage stability and performance of aqueous and dry formulation of Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) was conducted during 2011-2013 in Bio control laboratory, Department of Entomology and Insect Biotechnology laboratory, Biotechnology Centre, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola with an objective to develop potent HaNPV formulation for improving shelf life and performance. Aqueous and dry form of HaNPV formulations with antimicrobials, UV protectant and phagostimulant were prepared and studied their shelf life and performance during storage. Synthesized and characterized the Silver nanoparticles. Prepared fresh Ha NPV required for preparing different formulation. The contaminants associated, storage stability and larvicidal activity of formulated HaNPV was studied. Data revealed that, HaNPV formulations having Silver nanoparticles @ 8 μl/ml of HaNPV and 80 μl/ml of HaNPV checked the bacterial contamination up to 13 months of the storage period, did not affect the viability of POBs and insecticidal properties of HaNPV formulation. Aqueous form of HaNPV + Streptomycin @ 0.18 g/lit of HaNPV + Tinopal 1% + Sucrose 1% , dry form of HaNPV + Streptomycin @ 0.18 g/lit of HaNPV + Tinopal 1% + Sucrose 1% and HaNPV + Streptomycin @ 0.18 g/lit of HaNPV + sucrose remain stabled up to 12 months. However, aqueous form of HaNPV alone reduces the insecticidal properties from the 9th month of storage period. While, dry form of HaNPV alone reduces the insecticidal properties from the 12th month of storage period. From the result, it was noticed that dry form of HaNPV formulation having antimicrobial found more stable than aqueous form.

Influence of biological control agents Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV) on intensity of the attack twig caterpillar (Ectropis bhurmitra Wlk) and yield of tea plant (Camellia sinensis (L.) O. Kuntze) clone GMB 7

The objective of this research was to evaluate the effect of Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV) on attack intensity of twig caterpillar (Ectropis bhurmitra Wlk) and the yield of clone Gambung 7 tea plant. The research was conducted at the Research Institute for Tea and Cinchona, Gambung, Bandung, from May 2011 till June 2011. A randomized block design was used with five treatments and replicated four replications. Application of five controls method for the twig caterpillar were performed. HaNPV at  4 x 106 PIB/mI, and 4 x 104 PIB/ml, Bacillus turingiensis (Thuricide) 1 g/L, Spinosad (Tracer 120 SC) 0,5 ml/L, and chemical insecticides (Electric 500 EC) 0,5 ml/L. The results indicated that HaNPV at both the density gave the same effect compare to other insecticides tested on the attack intensity of twig caterpillar as well as yield of tea plant clone Gambung 7.

Influence of biological control agents Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) on mortality twig caterpillar (Ectropis bhurmitra Wlk.)

The objective of this research was evaluate effectiveness of Helicoverpa armigera Nuclear Virus Polyhedrosis virus (HaNPV) on twig caterpillar (Ectropis bhurmitra Wlk) mortality. The experiment was conducted at the Research Institute for Tea and Cinchona, Gambung, Bandung, elevation of + 1.300 m asl, from March 2011 till June of 2011. A completely randomized design was used with 12 treatments and replicated twice. Aplication of four level concentrations of HaNPV on three stadia of the twig caterpillar larvae were performed. The HaNPV concentrations were 4 x 106 PIB/mI, 4 x 104 PIB/ml, 4 x 102 PIB/ml and 0 PIB/ml, and stadia of the twig caterpillar larvae were 2, 3, and 4. The results indicated that direct application of HaNPV on twig caterpillar larvae had similar effect in mortality of the larvae, either in the laboratorium or in the greenhouse, with level of mortality was 70% within four to ten days periode. Laboratory expe­rimentation indicated that 4 x 102 PIB/ml concentration of HaNPV applied directly on stadia 3 and 4 was effective in suppressing Ectropis bhurmitra Wlk. larva population while for larvae stadium 2 the effective concentration was 4 x 104 PIB/ml. In greenhouse experiment revealed that 4 x 102 PIB/ml concentration of HaNPV was effective in suppressing larvae of stadium 2 and stadium 4, while until the end of the experiment no indication of maksimum mortality in stadium 3 population.

Biological Test of Formulation of Subculture Helicovepa armigera Nuclear Polihedrosis Virus (HaNPV) on Mortality of Spodoptera litura Larvae infested to cabbage (Brassica oleracea Var. capitata Linn.) Plantation

<p>Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV) is one of the biological agents that could be used for the development of organic farming in Indonesia. One of the organic agricultural food products which have high economic value is cabbage, HaNPV effectif to control the population of several species of insect pests, can be used instead of synthetic insecticides in agricultural development efforts of organic cabbage. HaNPV formulation can be used as a way to maintain the virulence properties of the virus and facilitate the storage and application. The preparation of subculture HaNPV formulations was in liquid, powders, cornstarch and talk carrier material. The design experimental was used randomized block design consists single factor (type HaNPV formulation consisting of powder formulations, liquid formulation, formulation in cornstarch, and talc formulation including water formulation without virus as a control), with eight replications. The results showed that all of the formulations of subculture HaNPV causing larvae mortality at range 97.4% -100%. The highest mortality of larvae occurred in cabbage plants are protected by HaNPV carrier liquid and talk preparations by 100% each. <br /><strong></strong></p><p><strong>Keywords</strong>: Formulations Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV), subculture, Spodoptera litura, Brassica oleracea Var. capitata Linn.</p>

THE BIOLOGICALTEST OF FORMULATION OF SUBCULTURE Helicoverpa armigera Nuclear PolyhedrosisVirus(HaNPV) against Crocidolomia pavonana Fab. LARVAE POPULATIONTHAT EXPOSED TO CABBAGE (Brassica oleracea var. capitata L.)

<p>The biologicaltest of formulation of subculture Helicoverpa armigera Nuclear Polyhedrosis Virus(HaNPV) against Crocidolomia pavonana Fab.larvae population that exposed to cabbage (Brassica oleracea var. capitata L.) has been done. The subculture HaNPV was formulated in liquid preparations, powders, cornstarch and talc carrier materials, sprayed to C. pavonana larvae population that exposed at cabbage as a pilot project. The method of research used an experimental method, with randomized block design consists of single factor HaNPV and five level formulation there were (p) (liquid dosage forms (p1), powder (p2), and mixed with a carrier such as cornstarch (p3) and talc powder (p4) as well as the provision of water control without virus (p0)) with 8 replications. The density of virus 4x107 polyhedral/ml. The results was statistically analyzed with ANOVA (p<0,05), showed that all formulation HaNPV subculture have equal ability to caused high mortality of C. pavonana larvae population. <br /><strong></strong></p><p><strong>Keywords</strong>: Cabbage, Crocidolomia pavonana, Formulation,HaNPV, Mortality</p>

THE POTENTIAL OF SUBCULTURE Helicoverpa armigera NUCLEAR POLYHEDROSIS VIRUS (HaNPV) TO BE UTILIZED AS AN ALTERNATE SYNTHETIC INSECTICIDES TO CONTROL INSECTS PESTS IN CABBAGES PLANTATION (Brassica oleracea var. capitata L)

<p>Subculture Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV) is an entomopathogenic viruses isolated from cadaver of Helicoverpa armigera larvae, and was succesfully produced in Spodoptera litura larvae as an alternate host. These viral agent have been applied to control the population of three species insects pests Spodoptera litura, Crocidolomia pavonana and Plutella xyllostella which were infested in Cabbages Plantation. The concentration of virus of 4 x 107 Polyhedral/ml was sprayed in cabbage leaves 24 hours before infested of larval and every four days after. The research used randomized design which consisted of factor (three species of second instar larvae, S. litura, C. pavonana, P. xyllostella) and eight replications. The result showed that the three species of larval were sensitive against subculture HaNPV infection. The mortality of each larval species (S. litura, C. pavonana and P. xyllostella) were 100%, 97.5% and 98.7%. Subculture HaNPV can be utilized to control the population of three spesies of insects pests in Cabbage Plantation. </p><p>Keywords : Subculture HaNPV, Polyhedral</p>

Ultraviolet protection of nucleopolyhedrovirus through microencapsulation with different polymers

A microencapsulated formulation of Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) was produced by emulsion technique to improve its stability under ultraviolet (UV) radiation. The polymers used include sodium alginate, gelatin and starch at concentration levels of 3% and 5% w/v. Except for the starch microencapsulated formulation (3% w/v), the difference in mortality of treated insects between microencapsulated and non-microencapsulated suspensions before irradiation was not significant according to variance analysis (Duncan test, P < 0.05, df = 6). This indicates that microencapsulation of HaNPV does not affect viral activity. Among the three polymers, gelatin performed the best and provided the most stable formulation. The Original Activity Remaining (OAR) percentage for the gelatin formulations did not change from its initial value after 24 h of irradiation. There was no significant difference between the OAR percentage values of 3% and 5% gelatin formulations after 72 h of UVA exposure (90 and 94, respectively; Duncan test, P < 0.05, df = 6). The OAR percentage for the gelatin microencapsulated formulation was 90 after 30 minutes of exposure to UVC radiation. However, for the non-microencapsulated virus suspension, the OAR percentage value declined sharply to 16 after 30 minutes of exposure to UVC radiation. Concerning the in vitro release behaviour of gelatin microparticles (MPs), virus release initiated quickly, but continued at a slower rate until it reached 100% after 1 h of exposure to the release media. The experimental data for the gelatin MPs showed good correlations with the Korsmeyer–Peppas semi-empirical model, indicating that the transport mechanism is primarily consistent with Fickian diffusion.

Biological control of cotton pests in China

Cotton is one of the most economically important crops in China, while insect pest damage is the major restriction factor for cotton production. The strategy of integrated pest management (IPM), in which biological control plays an important role, has been widely applied. Nearly 500 species of natural enemies have been reported in cotton systems in China, but few species have been examined closely. Seventy-six species, belonging to 53 genera, of major arthropod predators and parasitoids of lepidoptera pests, and 46 species, belonging to 29 genera, of natural enemies of sucking pests have been described. In addition, microsporidia, fungi, bacteria and viruses are also important natural enemies of cotton pests. Trichogramma spp., Microplitis mediator, Amblyseius cucumeris, Bacillus thuringiensis and Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) have been mass reared or commercially produced and used in China. IPM strategies for cotton pests comprising of cultural, biological, physical and chemical controls have been developed and implemented in the Yellow River Region (YRR), Changjiang River Region (CRR) and Northwestern Region (NR) of China over the past several decades. In recent years, Bt cotton has been widely planted for selectively combating cotton bollworm, H. armigera, pink bollworm, Pectinophora gossypiella, and other lepidopteran pest species. As a result of reduced insecticide sprays, increased abundance of natural enemies in Bt cotton fields efficiently prevents outbreaks of other pests such as cotton aphids. In contrast, populations of mirid plant bugs have increased dramatically due to a reduction in the number of foliar insecticide applications for control of the bollworms in Bt cotton, and now pose a key problem in cotton production. In response to this new pest issue in cotton production, control strategies including biological control measures are being developed in China.

Geographic variations and their impact on bioefficacy amongst Helicoverpa armigera Nuclear Polyhedrosis Virus isolates from India

Helicoverpa armigera Nuclear Polyhedrosis Virus (HearNPV) was isolated from the larva, obtained from four different geographic locations in India. All the HearNPV isolates appeared clear, irregular, six sided in outline with rounded edges; phase bright under phase contrast microscope; but varied in size, bioefficacy and restriction profiles. Anand isolate had largest mean diameter, highest frequency of large polyhedral inclusion bodies and gave highest total percent mortality with lowest LC50 and LT50 followed by Surat, Patancheru and Junagadh isolates. Mortality rate of Anand and Surat isolates projected two peaks at two different time intervals pointing at the presence of more than one genotype in the same host origin. Restriction digestion analysis produced many unique fragments for each isolates proving that variation existed between the four isolates. These studies have clearly demonstrated that baculoviruses are very diverse, and that this diversity occurs at a range of scales from between isolates collected from the same host species, to between caterpillars collected at the same site and, from the current study, within variants collected from within an individual larva. The efficacy of such geographic isolates can be greatly improved by a deeper knowledge of their physical, chemical and biological properties. The knowledge can be applied for strain selection of the viruses from wild populations which would prove useful to develop NPVs desirable for biopesticides.

Genetic diversity and differentiation of Helicoverpa armigera nuclear polyhedrosis virus isolates from India

India has a rich biodiversity of microbes. Soil is the major source for isolation of entomopathogens, after the infected insects themselves. Four isolates of Helicoverpa armigera nuclear polyhedrosis virus (HearNPV) were obtained from the samples collected one each from Anand, Surat and Junagadh of Gujarat and Patancheru of Andhra Pradesh. All the HearNPV isolates appeared as clear, irregular six-sided objects with rounded edges, phase-bright under phase contrast. Junagadh, Surat, Patancheru and Anand isolates gave 27.47–42.80%, 36.83–51.32%, 26.05–43.76% and 42.99–54.85% mortality, respectively, when the percent mortality was pooled over period. The least number of HearNPV polyhedral inclusion bodies (PIBs) (5.1 × 107) of the Anand isolate were required to kill 50% of the H. armigera population within 120 h. The Anand isolate was fastest (90.30 h), followed by Surat (120.26 h), Junagadh (139.53 h) and Patancheru (143.10 h) in killing 50% of the H. armigera population at a dose of 109 PIBs ml−1. RAPD analysis of all 15 arbitrary oligonucleotide primers generated 353 scorable bands with 201 loci. A total of 181 polymorphic bands were obtained, ranging in size from 141 to 1,873 base pairs. The percentage of polymorphic loci was 90.19%. The mean polymorphism information content (PIC) value for 15 primers was found to be 0.99. The similarity coefficient values based on 15 RAPD markers ranged from 0.235 to 0.407. The four isolates were grouped into two clusters: one cluster consisted of Junagadh and Anand and the second cluster consisted of Surat and Patancheru.