Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are obligate parasites of insects and are used as biological control agents of economically important insect pests. Genera Steinernema and Hetrorhabditis possess a symbiotic association with pathogenic bacteria from the Xenorhabdus and Photorhabdus genera, respectively [1]. EPNs are ubiquitous, as they have been found in a wide range of ecologically diverse soil habitats including cultivated fields, woodlands, grasslands, deserts and ocean beaches, except Antarctica [2]. Being insect’s natural enemy, having wide host range, host location searching / locating capability (particularly of some soil pest and stem borers) makes EPN a successful biocontrol agent [3]. Total 18 pesticide formulations, based on 12 different EPN species are commercially available worldwide as of now, whereas only 2 Steinernema carpocapsae formulations are available in India [4]. As potential natural enemies of insect pest, EPNs dominate its native habitat so there is the need of hour to investigate their entomopathogenic potential against major insect pests in India. Thus, the call for development of formulations using native EPNs has necessitates the search for new strains. Moreover, applying exotic EPNs may negatively affect native communities of EPNs and apparently dampen their rate of natural control [5]. Decrease in widespread EPN after application of exotic strain has been also reported with detrimental effects in long term [6]. Consequently, the isolation of native species of EPNs provides a valuable source for both biodiversity perspective and applicability prospect [7].

Infective juveniles (IJs), the only free-living stage of EPNs, enter the host insect through its natural apertures (oral cavity, anus and spiracles) or in some cases through the cuticle. Once inside the host insect, the nematodes and the multiplying bacteria in the hemocoel produce virulence factors resulting in insect death [8]. Developing nematodes feed on the bacteria by disintegrating host tissues, produce 1-3 generations and when the food resources are exhausted; nematodes emerge as IJs to seek new hosts [9]. During host invasion, the bacteria release several toxins and exoenzymes that play a role in insect death. The genome of Photorhabdus luminescens encodes a variety of virulence factors including toxins, hemolysins, adhesins, proteases and antibiotic-synthesis genes [10]. A number of insecticidal toxins from sp. have been reported viz., Tc toxin complex [11], mcf1 which is apoptotic to insect and mammalian cell lines [12], PVC (Photorhabdus virulence cassettes) similar to bacteriocins in P. asymbiotica [13] and the PirAB (Photorhabdus insect related) toxins which are similar to insect’s juvenile hormone esterase (JHE) of beetles [14].

The cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a polyphagous insect pest distributed over the world [15]. It causes an estimated loss of US$927 million in chickpea and pigeon pea, over US$5 billion on over 200 crop species belonging to 45 families worldwide [16] and over $1 billion (USD) annually on different crops in India alone [17]. Globally, the H. armigera caterpillar consumes up $5 billion each year as control costs and production losses. China and India devote 50% of their insecticide applications to controlling it. Even more alarming is the rise of Bt-resistant populations of H. armigera, which have been identified in Pakistan, China, India and Australia [18].

Spodoptera litura (Fabr.), the ‘tobacco cut worm’ is a ubiquitous, polyphagous, lepidopterous pest that feeds on 112 cultivated crops all over the world [19]. Its larvae normally feed on the tender leaves causing serious damage to a variety of crops such as tomato, chilies, banana, castor, ground nut, soya bean, winged bean and cocoa [20].

The present study was undertaken as an extension to the biocontrol potential study of native EPN isolates collected from different parts of India and to investigate their efficacy against major insect pests. Eight isolates were collected from different parts of India [21]. The preliminary study of virulence of all the strains was carried out on G. mellonella and the three best strains viz., Heterorhabditis sp. strain Hmg3 (accession no. FJ751864), strain Hms1 (accession no. HQ637414) and strain Hgj (accession no. FJ744544) were shortlisted for further studies. This paper reports the virulence of above mentioned three strains of Heterorhabditis sp. as well as symbiotic bacteria alone (Photorhabdus luminescens strains) to three lepidopteran pests viz., H. armigera, S. litura and G. Mellonella along with their reproduction potential.

Test insects

H. armigera and S. litura were reared in the laboratory on a chickpea based semi-synthetic diet as described by Kalia et al., Galleria mellonella was cultured on semi-synthetic diet as per [22]. Rearing environment of 27 ± 2°C temperature with 60 ± 5% relative humidity (RH) was maintained throughout the experiment. All the adult moths were offered 10% honey solution fortified with multivitamins during their egg-laying period, in addition G. mellonella and S. litura were also provided with butter paper and blotting paper folded in a fan shape respectively for egg laying in the mating jars.

Test entomopathogenic nematodes

Three Heterorhabditis sp. strain Hmg3 (accession no. FJ751864), strain Hms1 (accession no. HQ637414) and strain Hgj (accession no. FJ744544) were obtained from Dr. S. Ganguly, Division of Nematology, IARI, New Delhi and maintained at the Insect Physiology and Molecular Biology laboratory. All strains of EPN were cultured through last instar G. mellonella. The emerging infective juveniles (IJ) were harvested using White traps [23] and stored at 10-15°C until further use within 2 weeks. The Photorhabdus luminescens strains namely SG-MG3 (accession no. JX221722), SG-NGP (accession no. JX240394) and SG-GJ were isolated from strains Hmg3, Hms1 and Hgj respectively.

Bioassays with EPNs

The bioassays were performed on 12 well cavity sterile plates (2.5-cm-dia. x 2-cm-depths), where each well was lined at the bottom with Whatman No.1 filter paper. Hundred microliters of IJs suspensions were prepared in double distilled water (ddw) containing 10, 20, 50, 100 and 200 IJs of each isolates individually and incorporated onto the filter paper before releasing final instars larvae of H. armigera, S. litura and In the control 100 μl ddw alone was introduced onto the filter paper. Ten replicates per concentration were used and each treatment was repeated thrice. Incubation was performed at a constant temperature of 27 ± 2°C and 60 ± 5% RH. Mortality was observed at 12 h interval for first 48 h followed by 24 h interval up to 168 h of IJs inoculation. Corrected mortality was calculated by [24]. Median lethal concentration (LC₅₀) was calculated at 36 and 48 h as 100% mortality in G. mellonella was attained by 48 h. Median lethal time (LT₅₀) was calculated at different concentrations to evaluate the most efficient dose with minimum time.

Estimation of EPN production

Ten dead larvae from the bioassay experiment (under 2.3), were selected randomly and rinsed with ddw. The larvae were then transferred aseptically on to the White trap and each concentration had a single white trap with 10 replicates. The IJs were collected during a period of 30 days starting from the date of first harvest. The IJs suspension was collected in a 50 ml culture bottle and a constant volume of 30 ml was maintained for all the suspensions. For counting the IJs, three samples were collected from each suspension and a 25 μl aliquot was withdrawn from each one and transferred to a 7 cm nematode counting dish to be counted separately. Final nematode concentration per ml was calculated by multiplying the average of the three 25 μl counts.

Isolation and bio efficacy of symbiotic bacteria

Last instar larvae of H. armigera were exposed individually in sterile plates (5 cm diam. x 2 cm depth) lined with Whatman No.1 filter paper to 100 IJs of Heterorhabditis strains suspended in 100 μl ddw. After 24 h of exposure, fresh haemolymph was collected by making a lesion in a proleg, in pre-cooled centrifuge tubes. Ten μl of this haemolymph was streaked on NBTA plates (Nutrient agar 7; Bromothymol blue 0.025; triphenyl-2,3,4-tetrazolium chloride 0.04 g.l^-1) and was incubated at 28°C for 24 h. Pure colonies were purified by sub culturing thrice on NBTA plates and subsequently inoculated in 250 ml Luria broth. The inoculated broth was incubated at 28°C and 200 rpm for 24 h. Culture broth was centrifuged at 8,000 rpm for 15 min. at 4°C. Bacterial cell counts were made using ‘Neubaur Haemocytometer’ (Germany) and the number of bacterial cells/ml calculated. A stock of suspension of 1 x 10⁹ cell.ml^-1 was prepared.

Each P. luminescens strain was tested against neonates of H. armigera and S. litura by diet incorporation method using 100 μl suspensions maintaining the concentrations of 10, 1 × 10², 1 × 10⁴, 1 × 10⁶ and 1 × 10⁸ bacterial cells/gm of diet as per [22]. The control was incorporated with 100 μl ddw only. Each test was replicated thrice with 10 neonates per replicate. The mortality observations were recorded at every 24 h till 7 days.

Growth inhibition of H. armigera larvae after P. luminescens treatment

On 7^th day after treatment ten larvae from the above experiment were randomly selected from each set of concentration (treatment) and control. The percentage growth inhibition was calculated with the formula: Growth inhibition (%) = (C_w-T_w)/C_w) × 100, where C_w is the average weight of 10 larvae in control and T_w is the average weight of 10 larvae in treatment.

Statistical analysis

The insect mortality data for median lethal concentration (LC₅₀) and median lethal time (LT₅₀) were analysed using maximum likelihood program for probit analysis [25]. The LC₅₀ and LT₅₀ were considered significantly different only in case of non-overlapping fiducial limits at 95% confidence level. The IJ production analysis was performed by one way analysis of variance (ANOVA) (SAS 9.2, SAS Institute Inc., Cary, NC, USA). Correlation between the parameters was determined by regression analysis.

Bioassays with Heterorhabditis strains

Perusal of data in (Table 1) revealed that Hgj strain was observed to be most virulent against G. mellonella with lowest LC₅₀ at 36 h (13.02 IJs/larva) followed by S. litura (LC₅₀ = 23.49 IJs/larva) and H. armigera (LC₅₀ = 115.24 IJs/larva). However, all the three strains were found to be at par against G. mellonella at 36 h with LC₅₀ values ranging from 13.02 to 14.58 IJs/larva and overlapping fiducial limits exhibiting no significant difference among them. By 48 h strains Hms1 and Hgj demonstrated 100% mortality, in contrast LC₅₀ for Hmg3 was 7.59 IJs/larva only. On contrary, H. armigera was observed to be least susceptible to Hms1 with highest LC₅₀ (138.56 IJs/larva) followed by Hgj (LC₅₀ = 55.52 IJs/larva) and Hmg3 (LC₅₀ = 36.01 IJs/larva) at 48 h. In case of S. litura, Hgj (LC₅₀ = 23.49 IJs/larva) was yet again the most effective strain with least mortality at 36 h followed by Hms1 (LC₅₀ = 72.03 IJs/larva) and Hmg3 (LC₅₀ = 98.50 IJs/larva). But, Hmg3 required only 10 IJs per larva by 48 h, followed by Hms1 and Hgj (LC₅₀ = 12 and 14 IJs/larva respectively).

Table 1: Toxicity of three strains of Heterorhabditis sp. against last instar Helicoverpa armigera, Spodoptera litura, and Galleria mellonella.

LT₅₀ values of the strains vs. test insects were found to be rate dependent with IJ dose (Table 2a). LT₅₀ values in G. mellonella ranged from 19.23 h to 45.89 h which was also the narrowest range among the test insects. LT₅₀ of Hmg3 at 200 IJs was observed to be 19.23 h (least among all the doses as well as all the strains) and was significantly different from 10 and 20 IJs (38.87 h and 34.84 h respectively)(r = -0.876). While Hms1 and Hgj showed similar LT₅₀ (20 h) at the dose of 200 IJs which was significantly different from 10 IJ dose (Hms1 LT₅₀ = 45.84 h and Hgj LT₅₀ = 45.89 h) towards G. mellonella.

Table 2a: LT₅₀ values calculated from dosage response assays conducted with three strains of Heterorhabditis sp. against last instar Galleria mellonella.

In case of H. armigera as well, Hmg3 showed least LT₅₀ (32.34 h) at the dose of 200 IJs followed by Hgj and Hms1 with 40.96 h and 44.08 h LT₅₀s respectively on the same dose (Table 2b). None of the strain was found to attain 50 % mortality at the 10 IJs concentration till 168 h against H. armigera. However LT₅₀ @ 200 IJs for Hgj was found to be significantly different from 50 IJ concentration, whereas the other two strains were found to be at par (Table 2b).

Table 2b: LT₅₀ values calculated from dosage response assays conducted with three strains of Heterorhabditis sp. against last instar Helicoverpa armigera.

Lowest LT₅₀ of 33.35 h was observed with strain Hgj against S. litura followed by Hms1 (LT₅₀ = 40.02 h) and Hmg3 (LT₅₀ = 56.24 h) at the 200 IJs, although found to be at par at these concentration (Table 2c). While 200 IJs in Hgj was significantly different from 10, 20 and 50 IJs treatment with in the strain, it was found to be significantly different from 10, 20 50 and 100 IJs treatment among the rest two strains within the test insect.

Table 2c: LT₅₀ values calculated from dosage response assays conducted with three strains of Heterorhabditis sp. against last instar Spodoptera litura.

In case of G. mellonella, for 10 fold decrease in the IJ concentration (10:100) 1.7-1.9 fold increase in LT₅₀ was observed in among all the test strains. However in H. armigera, none of the strain able to attain 50% mortality @ 10 IJs till 7 days after treatment but in term of LT₅₀, all the strains were found to be at par with the G. mellonella at the highest concentration tested (200 IJs). Strain Hgj was most effective against S. litura in terms of infectivity with 3.4 fold decline with 10 times increase in IJ dose. While Hmg3 and Hms1 strains showed only 1.5 and 1.7 fold decline respectively. But in term of LT₅₀, all the strains were found to be significantly different with respect to G. mellonella at the highest concentration tested (200 IJs). In general for Heterorhabditis strains vs. the test insect analysis, LT₅₀ was found to be negatively correlated with the rate of application.

Estimation of IJ yield

Perusal of (Table 3) Hms1 (Nag) was the most effective strain for progeny production and yielded 160.38 × 10³IJs /larva in G. mellonella and 190.39 × 10³ /larva in H. armigera at the initial dose of 200 IJs/larva.

Table 3: Production of three strains of Heterorhabditis strains IJs in last instar Helicoverpa armigera, Spodoptera litura, and Galleria mellonella.

In G. mellonella lowest yield was 56.39 × 10³ /larva, obtained in strain Hms1 at the dose of 10IJs/larva. IJ Yield in strain Hms1 at 10 and 20 IJ/larva (56.39 × 10³ and 64.33 × 10³ respectively) and 50 and 100 IJ/larva (109.08 × 10³ and 118.94 × 10³ respectively) was found to be at par (F_4,14 = 246.85, p<0.0001). Second best IJ yield at the initial dose of 200IJs/ larva was observed in strain Hgj (139.01 × 10³ /larva), which was significantly different from the yield among the other doses of this strain. It was also observed that production at 100IJs and 20IJs were significantly different from each other but both were equivalent to 50IJs. Least productivity for the strain Hgj was observed at the dose of 10IJ (97.49 × 10³ /larva), which was significantly different from rest of the doses (F_4,14 = 2087.1, p<0.0001). Lowest production at the dose of 200IJs/Larva (124.10 × 10³ /larva) was observed in strain Hmg3. Although a positive correlation (r =0.940) between the increase in concentration and the IJ production was observed, however there was a significant difference between 10, 20, 50 and 100 IJ/larva doses for this strain(F_4,14 =370.46, p<0.0001).

In H. armigera, the IJs production was observed to be in the range of 40.65 x 10₃ /larva (10 IJ/larva dose) to 190.39 × 10³ /larva (200 IJs per larva) with Hms1 as the most efficient strain for IJ yield. In Hms1 all the concentrations were positive correlated (r = 0.952) to the IJ yield but were significantly different from each other (F_4,14 = 1125.81, p<0.0001). Hmg3 is the second best strain for production of IJs with 164.82 × 10³ / larva at the concentration of 200IJs, followed by Hgj with 160.63 x 10³ / larva at the same dose. Strain Hmg3 yield at 10 and 50 IJs (40.65 x 10³ / larva and 49.27 × 10³ /larva respectively) is significantly different from each other but at par with 20 IJs (42.45 × 10³ /larva), while production at 100IJ is different from rest of the concentrations (F_4,14 = 2559.84, p<0.0001). Although the relative yield at the concentrations of 10, 20 and 50 IJ for Hmg3 had been lowest as compared to the relative production from the prior mentioned concentrations of the other two strains. In contrast, the IJ reproduction levels in strain Hgj had been observed to be moderately good at the concentrations of 100 (133.44 × 10³ /larva) and 50IJ (103.69 × 10³ /larva) which were significantly different from each other as well as from rest of the concentrations (F_4,14 =76.83 p<0.0001).

The ratio of yield per dose was calculated to evaluate production maxima from smallest dose of inoculum. With G. mellonella as the host for the in vivo IJ production the strain Hgj was observed to be the best with the ratio of 6.75 × 10³ yield/10IJs, followed by 6.05 × 10³ yield/10IJs in strain Hmg3 and 5.64 × 10³ yield/ 10IJ (significantly different from each other). While in case of H. armigera, Hms1 was the best strain with 7.37 × 10³ yield/ 10IJs which is found to be statistically at par with Hgj having 6.92 × 10³ yield/10IJs.

Based on the above results in terms of IJ production as well as yield/inoculation ratio Hms1 is the most efficient strain in H. armigera. On contrary, in G. mellonella Hms1 is best for production while Hgj resulted in highest yield/inoculum ratio. Production of IJ was positively correlated with the IJ concentrations for all the three strains in both H. armigera and Though, yield/ inoculum ratio was negatively correlated with the concentrations.

Isolation and bioefficacy of symbiotic Photorhabdus luminescens strains

Feeding assays of symbiotic bacteria Photorhabdus luminescens to S. litura neonates, revealed P. luminescens strain SG-Ngp as most effective by day 4 with 3.74 × 10⁶ cells/ gm of diet (Table 4). While P. luminescens strain SG-Mg3 was also effective with 8.49 × 10⁷ cells/ gm of diet, P. luminescens strain SG-gj was observed to have 36.67% mortality even at the highest concentration tested (i.e. 2 × 10⁸). A decrease in LC₅₀ was observed at day 7 in all the strains, although SG-Ngp strain (LC₅₀ = 4.06 × 10⁵ cells/ gm) remained the most effective against S. litura. Strain SG-Mg3 was moderately effective (LC₅₀ = 2.96 × 10⁶ cells/ gm) while SG-gj being least effective among the three (LC₅₀ = 2.93 × 10⁷ cells/ gm) against S. litura.

Table 4: Efficacy of the three Heterorhabditis symbiotic bacteria Photorhabdus strains against neonates of Helicoverpa armigera and Spodoptera litura after 4 and 7 day of treatment.

It is noteworthy that the three strains showed lower LC₅₀ against S. litura compared to H. armigera which concurrent with the results of IJ experiments.

Growth inhibition of H. armigera larvae after P. luminescens treatment

Perusal of (Table 5), a dose dependent inhibition in growth was observed in the 7^th day larvae of H. armigera. The average weight of the larvae in control at day 7 was 41.7 mg. A decline of 20-31% was observed in the larval growth at the dose of 10¹ P. luminescens cells/gm of diet. While average larval weight of ~23 mg was observed at the concentration of 10² cells/ gm of diet (43% growth reduction) for Hgj and Hms1. Hgj was able to reduce larval growth up to 13 mg (68%) while 7.4 mg (82.25%) was the average larval weight in Hms1 treatment of 10⁶ cells of the respective strains. Highest growth arrest of 98% was detected in Hms1 (F_5,58 = 39.14, p<0.0001) at the dosage of 10⁸ while at the same dosage 83% growth inhibition was exhibited byHgj (F_5,58 = 14.09, p<0.0001) treated larvae.

Table 5: Growth Inhibition in H. armigera.

The results obtained in the present study evidently showed that the virulence of the three strains of Heterorhabditis sp. tested to model insect G. melonella vis a vis polyphagous insects H. armigera and S. litura varied considerably, thus suggesting that each strain presents diverse virulence degrees in terms of LC₅₀ as well as LT₅₀. This is substantially documented in literature [26-28]. However, the dosage of EPNs remain crucial, as a dosage that is too low results in low host mortality and a dosage that is too high may result in failed infections due to competition with secondary invaders [29]. Thus, LC₅₀ values support in determining IJ dose for a particular insect host. Strains used in the present study have promising insecticidal action against H. armigera and S. litura at 48 h based on lower LC₅₀ as well as LT₅₀ values [30,31].

Similar to present results, [32] reported that the median lethal time was negatively correlated with increase in H. indica dose. They also reported the LT₅₀ of the laboratory assay on G. mellonella, H. armigera and S. litura (36 h, 40 h and 48 h respectively), which deduces S. litura as the sturdiest of the three insects. In contrast, present study reported G. mellonella to be most susceptible for all the three strains, while strains Hms1 and Hgj exhibited lower LT₅₀ in case of S. litura as compared to H. armigera which concurs LC₅₀ and LT₅₀ interpretations of [33,32]. Thus, Hms1 is the most effective strain against S. litura, whether provided in IJ form or as symbiotic bacteria alone.

Besides, infectivity and mortality, mass production of IJs is also considered as an important criterion to assess EPN efficiency. Poor reproduction of EPNs may hamper their cost effectiveness in largescale production systems [34]. In general nematode yield depends up on host size, nematode dosage and host density [35,26]. Several Heterorhabditis sp. had been reported to have in vivo production ranging from 8.0 × 10⁴- 5.67 × 10⁵ IJs per larva using G. mellonella as the host [36,35,28]. Our result validates these findings as the average yield of the three Heterorhabditis strains was found to be in this range.

Among the three strains, Hms1 was most efficient IJ producer, using both G. mellonella and H. armigera. Although, when H. armigera was used as the host for this strain, the yield increased by 16%. It has been reported that, the quality as well as the composition of the lipids in the host insect play a major role in the production and yield of infective juveniles [37]. average IJs production using the same host in Steinernema sp. had been reported in 71 × 10³ IJs per ml (S. feltiae) to 320 × 10³ IJs per ml (S. carpocapsae) [38,39], which is lesser as compared to Heterorhabditis. The reproductive potential of Heterorhabditis strains was observed to be higher as compared to S. thermophilum at the same dosage range [22] Kalia et al., which concurs with the results of [40].

Photorhabdus species are known to be highly virulent towards a wide range of insect hosts. While there are several reports of both injectable as well oral activities of its purified toxin complex, however reports on oral toxicity of P. luminescens bacteria alone remain insufficient. As discussed by [22], using the symbiotic bacteria alone is a promising potential avenue for biological control, particularly because the bacteria are less expensive to produce than the nematode-bacteria complex. Oral toxicity on P. luminescens and X. nematophila against Aedes aegypti larvae was found to be 83% and 52 % respectively [41]. In addition, the EPN bacterial symbiont species culture suspensions have been used as immunosuppressant against Aedes albopictus and Culex pipiens pallens, along with B. thuringiensis [42]. Xenorhabdus sp. and Photorhabdus temperate subsp. temperata bacteria have been reported to cause high mortality of third-instar larvae of Spodoptera exigua, but not to the fifth-instar larvae when administrated orally [43]. The strains SG-NGP and SG-GJ, used in this study do exhibit growth dependent oral toxicity for the both H. armigera and S. litura, however SG-MG3 does not exhibit a dose dependent mortality for H. armigera.

One of the important aspect of this study is the reduction in average larval weight gain in both H. armigera as well as S. litura upon administering the two P. luminiscens (i.e. SG-NGP and SG-GJ) strains mixed at various concentrations in semi-synthetic diet. Several studies suggest restrained growth upon administration of EPN toxin in host [44]. Reported 10 strains of entomopathogenic bacteria exhibited over 75% antifeeding activity in 2^nd instar larvae of diamondback moth, Plutella xylostella by using leaf-disc test. However, current study highlights the dose dependent effect of the SG-NGP and SG-G on both lepidopteran insects. This growth inhibition may have resulted from disruption of normal physiology of the insects due to various proteins as well as non-protein toxins for their potential insecticidal or growth inhibitory effects reported from EPN associated symbionts [45]. A 48kDa protein, Txp40, has been reported in 58 strains of Photorhabditis and Xenorhabdus sps., recombinant form of this protein caused cell growth inhibition in vitro cytotoxicity assay of Aedes aegypti cells [46]. Another study describes a 63kDa protein from P. luminescens having growth inhibition action towards Manduca sexta [47]. These studies suggest EPN associated symbionts to be effective feeding deterrents. Strain SG-MG3, however lacks growth inhibition effect in the two host insects. This study provides an important insight on the native EPN strains with possible insecticide potential and may be an addition to the prevalent pest management strategies. Further our studies suggest that not only EPN but also its associated symbiotic bacteria alone can be used for effective pest control.

The authors acknowledge World Bank-funded NAIP-ICAR project (IARI- 70:13) for sponsoring this research project. We are grateful to Director, Indian Agricultural Research Institute, New Delhi, for providing infrastructure and facilities for carrying out these studies. The manuscript is dedicated to the memory of Dr. (Mrs) Sudershan Ganguly who was the Project coordinator of the aforesaid project.