Department of Botany

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Open Access
Application of essential cationic micronutrients as nano-fertilizer on vegetation propagation and growth of mulberry
(University of North Bengal, 2024) Salman Haydar; Roy, Swarnendu
Mulberry is a high biomass-producing, fast-growing, perennial plant belonging to the genus Morus under the family Moraceae. Mulberry leaves, especially those of the Morus alba L. (white mulberry), are agriculturally more important, serving as the only food source for the monophagous insect Bombyx mori (silkworm). It is estimated that almost 90% of global raw silk production depends upon mulberry silk, and in India, mulberry silk culture is performed mainly by moriculture (mulberry plant culture). Factually, India is the second largest producer of silk, and this silk industries have an immense contribution to its national economy. India's textiles and apparel industry holds economic importance, contributing 2.3% to the GDP, with a 12% share in exports and providing direct employment to 45 million people, along with 100 million in allied industries. Beyond its role in the sericulture industry, these plants have several medicinal properties, offering a range of health benefits, have strong food value, and serve as a component for phytoremediation purposes and landscaping practices. Despite its numerous benefits, the cultivation area for mulberries is constraining both globally and in India. Due to rapid urbanization, expanding cultivable land is quite difficult, so improving the productivity of mulberry foliage can meet the growing demand for silk. In addition to limited land availability, the high necessity of water and scarcity of proper water resources chronically affect the mulberry plantations. Lack of adequate nutrient supply is another prime factor inhibiting the mulberry growth and development. The cultivation of mulberry begins with the planting of saplings. In the case of mulberry, the preferred method for proliferation is through vegetative cuttings, as it proves to be the most efficient practice for the mass production of saplings. However, this process has shown significant delays in the succession of cuttings, the formation of roots, the flourishing of buds, and subsequent development into leaves, eventually delaying the leaf harvesting time. Here, adequate water and nutritional elements were also found to be two important factors determining the success rate of this propagation process. Conventionally, chemical fertilizers were employed in the nutrient management process, but they have several adverse effects on the environment and human health. While biofertilizers show promise as an alternative, they are condition-specific and effective only in certain soil conditions. Recently, nanotechnology-based nano-fertilizers have emerged that are reported to play a potent role in both nutrient management and abiotic stress amelioration. Concerning all these factors, in this study, iron, zinc, manganese, and copper nanoparticles (FeNP, ZnNP, MnNP and CuNP, respectively) were green synthesized using the aqueous decoction of pruning litter of tea plants [Camellia sinensis (L.) Kuntze] is a waste product of the tea industry and is widely available across North East and South Eastern India. During the synthesis, variation in the preparation process was followed to ensure minimal use of metallic precursor and plant extract and to prescribe an ideal synthesis condition. This involved mixing of 10 mM FeCl3, 100 mM of Zn(NO3)2, 500 mM NaOH, 1 mM KMnO4, and 500 mM of Cu(NO3)2 with 100 mg/mL extract of tea leaves while maintaining a volumetric ratio of 10:1 (metallic precursor: tea extract) for FeNP, CuNP and MnNP. Similarly, for ZnNP, the optimal ratio was 7:2:1 for zinc nitrate, NaOH, and tea extract, respectively. Notably, variations in light intensity, reaction time, temperature, and pH of the plant extract did not significantly affect nanoparticle synthesis, although extreme values should be avoided. All the synthesized nanomaterials showed a round to irregular morphology with a size range of 100 nm, as observed through instrumental characterization. The nanoparticles formed under optimum conditions, were then applied to mulberry vegetative cuttings in different concentrations to determine the effectiveness of the applied nanoparticles in mulberry growth and propagation. The results demonstrated that FeNP and ZnNP applied at 10 mg/kg soil dosage and MnNP and CuNP applied at 5 mg/kg soil dosages were found to be optimum. These treatments significantly enhanced the sprouting percentage, survival percentage, and various phenotypic characteristics of the studied plants. Subsequently, phytotoxicity analysis through Allium cepa root tip bioassay confirmed that the optimum dosages for mulberry growth were safe and did not possess any potential cytotoxic and genotoxic effects. Combining all the studied micronutrients is more efficient than the nanoparticles applied individually. This combined application effectively minimizes the adverse effects of a sixday watering gap, enhancing the viability of vegetative cuttings and facilitating subsequent plant growth as evaluated under drought conditions. The nutrient mobilization study indicated a substantial increase in soil micronutrient levels after nanoparticle application. Beyond the soil, this treatment significantly heightened the absorption, uptake, accumulation, and translocation of applied micronutrients within the plant system, as evidenced by bioaccumulation and translocation studies. As the silkworm is the ultimate consumer of the mulberry leaves, a silkworm rearing practice was carried out by feeding the nano-micronutrients treated leaves to ensure the safety concerns of these nanoparticle treatments. Larvae supplemented with the leaves treated with a combination of all the essential micronutrients demonstrated enhanced ingestion and digestion, along with improved absorption and assimilation of nutrients, ultimately resulting in an increase in larval body weight. The findings from the current study also reveal that leaves treated with combined treatments do not induce larval mortality but instead positively impact enhancing silkworm growth parameters. Though nanoparticles showed all the possibilities in plant nutrient management, bulk delivery of nutrients and nutrient leaching problems are the major concern. Further, there is still enough scope to improve the nutrient use efficiency (NUE) of these nanofertilizers. Slow and controlled release nano-fertilizers (SNRF and CRNF) are one step ahead that can solve the mentioned problems more efficiently. These are the coated or composite nano-fertilizers that release nutrients in a controlled and regulated manner. In this study, we synthesized four different kinds of SRNFs, taking hydroxyapatite, chitosan, graphene oxide, and hydrogel as the matrix polymer. In all the SRNFs, Fe, Zn, Mn, and Cu are loaded, and the prepared SRNFs demonstrate efficient nutrient loading and encapsulation efficiency. The prepared SRNFs also demonstrated satisfactory nutrient holding capacity and showed slow release of nutrients over a prolonged period of time. Furthermore, as evidenced by morpho-biochemical analysis, all the synthesized SRNFs were very effective in improving mulberry growth, propagation, and internal health. However, among all, hydroxyapatite-based SRNFs had the highest efficacy and also it stood to be first in terms of NUE. This comprehensive approach aims to ensure precision in micronutrient management for mulberry. Regardless of the possible benefits of these nano-fertilizers, their incorporation into plant systems, the specific transportation mechanism, and the ultimate status of uptaken nanoparticles inside plant systems should be investigated.
Open Access
Studies on the selected tea garden weeds for the promotion of tea plant health
(University of North Bengal, 2024) Ghosh, Sandipan; Sen, Arnab; Ghosh, Chandra
Tea, cherished worldwide as a beverage of choice, holds a dominant position in India's agricultural landscape, representing 75% of the country's tea production. The North East region of India holds immense importance, making a substantial contribution of approximately 75% to the overall output. However, despite its paramount importance, tea production faces numerous challenges, chief among them being pest attacks. These attacks pose a significant threat, often leading to substantial losses that can range from 30% to 50%. Among the array of pests, the Red Spider Mite (RSM) poses a significant threat, characterized by a short life cycle and prolific reproductive capacity. In North East India, the economic threshold level for RSM is identified at 5 mites per leaf. The persistent use of conventional pesticides, coupled with the mite's rapid life cycle, has fostered considerable pesticide tolerance, leading to recurrent and challenging resurgences, thereby impacting the Indian tea trade. The tea plant is susceptible to diverse pests, including tea mosquito bugs, red slugs, red spider mites, thrips, jassids, etc. Among these, red spider mites and tea mosquito bugs stand out as predominant pests in the region, causing substantial crop losses in the tea industry annually. The association of Oligonychus coffeae with tea dates back to the inception of tea production in Assam, North East India, in 1868. While Nietner initially discovered O. coffeae on coffee plants (Coffea arabica) in Sri Lanka in 1861, it was Wood -Mason who, in 1884, designated it as a new species, Tetranychus bioculatus. However, Pritchard and Baker's 1955 studied on the family Tetranychidae prompted a revaluation, leading to the recognition of T. bioculatus as a synonym of the coffee mite, eventually placing it under the name Oligonychus. According to early data, the red spider mite, Oligonychus coffeae Nietner, (Acarina: Tetranychidae), is one of the most significant pests of Indian tea, and causing up to 35–40% of the crop to be lost. Red spider mites and tea mosquito bugs are seasonal pests. The former occurs during the summer months and the latter during rainy winter. RSM is a polyphagous pest that feeds on coffee, rubber, indigo, grape, cashew nut, citrus, mango, Camellia, camphor, mulberry, oil palm, and many other tropical plants. It normally infests the upper surface of mature tea leaves, feeds along the midrib and veins, and gradually spreads to the entire surface of the leaf, thereby changing the color of the leaf to ruddy bronze. Larvae, nymphs, and adult mites wreak havoc on the tea plant by feeding on the sap of the leaves and sporadically on the petioles. In severe infestation, it damages the younger and older leaves and ultimately leads to defoliation and debilitation of the tea bush causing a crop loss of 14–18%. In South India, the economic threshold level (ETL) of RSM in tea is reportedly 4 mites per leaf. and 2–3 mites/cm2 in North EastIndia. However, ETLs vary from area to region and even from field to field due to crop phenology, the cost of pesticides and manpower, weather patterns, etc. The RSM lives under the cover of a silken web spun on the leaf surface as protection against inclement weather. Leaf temperature and light penetration within tea bushes also influence mite distribution; O. coffeae prefers the middle zone of the bush (30 cm below the plucking surface) because of optimum temperatures associated with plant shading. The temperature in the upper zone of the tea plant may reach 40– 45 0C yet shading can bring down the temperature of the middle tier of a bush to ambient levels of 30–32 0C. Tea plantations use synthetic pesticides to manage pests, but researchers need to explore plants' bio pesticidal properties for environmental problems. Native plant materials like oils, extracts, and secondary metabolites protect against pests and illnesses. Plant-based preparations, including powders, solvent extracts, essential oils, and insecticidal properties, are also used as biopesticides. Botanicals play a vital role in organic tea production by effectively controlling pests such as red spider mites and keeping their population below the Economic Threshold Level (ETL). Consistent use of plant extracts from the same species mitigates herbivore responses and minimizes the risk of pest resistance development. Numerous studies from previous literature affirm that plant materials are abundantly accessible within local regions. The utilization of crude or raw plant extracts, containing a diverse amalgamation of bioactive components, emerges as a straightforward and economically feasible solution. This approach, exemplified by the utilization of aqueous extracts derived from various indigenous plants in India, underscores the practicality and cost effectiveness of harnessing natural resources for various applications. Plant based remedies for the red spider mite have included Clerodendron infortunatum L., Acorus calamus L., Aegl e marmel os (L.) C orrêa, Xanthium strumarium L., Terminalia chebula Retz., Duranta repens L., and neem kernel. The selection of plants should not limit the available plants to guarantee a wide range of protectants, and ongoing research into more potential plants is encouraged. It is significantly more important to carry out multiple bioassays when evaluating botanical pesticides. So objective of our study is the survey and documentation and characterization of plants used in preparing bio formulation for managing the pest. In different tea estates of tarai and dooars region and tea research institute start to introduce some cultural practices in combating tea pest using some locally available weed plants. With such information in together, we chose ten weed plants based on the results of the survey data. Then collect those plant and prepare a bioformulation. We also prepared individual plant aqueous extracts to know which plant is more effective than a formulation and found Clerodendron infortunatum L., Artemisia vulgaris L., and Justicia adhatoda L. confer better results than other plants when applied to a red spider mite in a laboratory condition. Next, we formulate bioformulations using locally sourced plants, drawing upon both existing literature and the traditional knowledge of tea garden inhabitants. Initially, to evaluate the effectiveness of these bioformulations, greenhouse experiments were conducted, administering them at different dosages to infected samples. Following the acquisition of promising outcomes, we advanced to field trial experiments carried out in two distinct tea estates, situated in both the Terai and Dooars regions. As part of our ongoing exploration into the composition of the bioformulation, we embarked on comprehensive GCMS analysis, revealing the presence of specific compounds within its formulation. To gain deeper insights into the potential impact on insect pests, particularly the two-spotted red spider mite (Tetranychus urticae), we selected relevant proteins from the PDB database. Employing molecular docking studies, we substantiated that the presence of these identified compounds in the bioformulation could be a contributing factor to the mortality of the insect pest. Additionally, our findings are in line with earlier literature studies that underscore the pesticidal activity linked to these compounds. In the metagenomic approach applied to the bioformulation, an intricate exploration of the microbial flora within the bioformulation was unveiled. Furthermore, the reverse ecology study lends support to the notion that there exists a robust interaction among the bacteria, influencing their impact on the pest population. Upon inflicting damage to the pest, the ensuing generation of reactive oxygen species (ROS) triggers the activation of antioxi dantenzymes. Our investigation involved the collection of leaves from both infected plants and those treated with the bioformulation, alongside a positive control. Subsequent analyses, including various enzymatic and nonenzymatic tests, revealed that our bioformulation contributes to the reduction and mitigation of biotic stress. Moreover, our bioformulation demonstrated a positive influence on the activity of Plant Growth-Promoting activity by inducing changes in soil physiochemical composition. This, in turn, resulted in improvements in plant height, leaf count, and flushes, showcasing the multifaceted benefits conferred by our bioformulation.
Open Access
Ethno-medicinal and molecular profiling of fermented beverages consumed by different tribes in North Bengal, India
(University of North Bengal, 2024) Bhattacharjee, Soumita; Sen, Arnab; Ghosh, Chandra
The widespread adoption of preserving food through natural fermentation has ancient origins, primarily stemming from the ancestral wisdom of Tribal communities. Ethnic foods and beverages are described as culinary offerings that have their roots in the heritage and culture of a particular ethnic group, drawing upon their understanding of locally sourced plant and animal ingredients. India houses a multitude of tribal communities spread across diverse geographical regions. Among these communities, alcoholic beverages hold considerable popularity among the tribal population. As a preservation technique, the ancestral practice of natural fermentation has endured through the indigenous knowledge transmitted by ancestors within these tribes. The mildly alcoholic, sweet-flavoured beverages are regarded more as food than alcoholic drinks due to their highcalorie content, vitamins, and beneficial lactic acid bacteria and yeast. Traditionally prepared alcoholic beverages are cereals such as rice, wheat, maize, or ragi, which have been the substrate of choice for their preparation, depending on the habitat of the tribal population. For generations, alcoholic beverages have been consumed at diverse cultural, social, and religious events. Most fermented drinks predominantly use cereals as a base, incorporating many plants and fruits as the primary raw materials. This combination contributes to a distinct and unique taste in the beverage. Numerous traditional ethnic practices have been ignored, resulting in insufficient documentation for scientific analysis. Therefore, the present investigation was conducted to evaluate the appropriate scientific examination of select tribal beverages in North Bengal, India. Initially, various areas inhabited by tribal communities were selected to gather their respective traditional beverages, used plant parts and the starter culture. The initial screening was based on the alcohol content of the beverages. Eu, a millet-based drink utilized by the Toto tribe in North Bengal, Chokot, a ricebased drink consumed by the Rabha tribe in North Bengal, Harchur raksi, and Timboor raksi, traditional medicinal plant-based drinks used by the tribal community in the highaltitude region in Darjeeling Himalaya, and Haria, a rice-based drink consumed by the Orao and Santal communities in North Bengal, were selected for further study. The injection of starter cultures, serving as the source of ethnic microbiota to induce the fermentation process, was also included in the present study. In vitro, the antioxidant study was also conducted to evaluate the most potent among those, as mentioned earlier, low-alcoholic ethnic drinks. The study results show that Eu is the most potent beverage among the other tribal drinks. The Harchur raksi (HR) is a lesserknown beverage consumed by the indigenous people residing in the high -altitude regions of the entire Himalayan range. HR is unique in its traditional processing method, an indigenous knowledge held by the Darjeeling-Himalayan tribal community. This knowledge should be meticulously documented to preserve its uniqueness. Although HR is considered an anti-inflammatory brew with various ethnomedicinal claims by the local inhabitants, there has been no scientific evaluation thus far. A comprehensive survey was conducted across the Darjeeling Himalayan region to unearth the ethnobotanical knowledge related to HR. HR was identified and collected in collaboration with a local expert. This work aims to assess the physical, biochemical, and metabolite profiling of HR using different biochemical tests and Gas Chromatography–Mass Spectrometry (GC–MS) experiments. To get a deeper understanding of the medicinal properties of HR, in silico molecular docking studies were conducted. The analysis revealed that HR, with a notably low alcohol content (around 6–8%), exhibits high antioxidant activity. GC–MS analysis of the brew identified 27 bioactive compounds, which were further subjected to bioinformatics analysis. Molecular docking analysis demonstrated the inhibitory activity of most of these compounds against oxidative stress, inflammation, and specific potent viral proteins related to their pathogenicity. Timbur raksi is a distilled fermented alcoholic beverage from the highaltitude region of Darjeeling Himalaya. The methanolic extract of the sample exhibited 60% inhibition at a higher concentration (200μg/ml). The GC-MS evaluation of the sample revealed the presence of 43 bioactive compounds, which have potential applications as scavengers for reactive oxygen species, anti-inflammatory agents, and anticancer compounds. 'Choko'/'Chokot' is a rice-based beverage consumed by the ethnic tribal community 'Rabha' in North Bengal and Assam, India. While rice is the main ingredient in this fermented product, introducing the starter culture, popularly known as 'Bakhor or phap,' brings variation to its composition. 'Choko'/'Chokot' constitutes a significant portion of the daily caloric intake for the Rabha tribe. Metabolite profiling was examined through GC-MS analysis. Additionally, the Metagenomic analysis was employed to assess the total microbial load of the starter culture (Bakhor). Reverse ecology analysis delved into the interaction between the microbes present in the brew and humans. The results indicated that the compounds in this beverage mostly exhibit antimicrobial and antifungal effects despite its relatively higher alcohol content than other tribal ethnic drinks. The metagenomic analysis identified several dominant bacterial genera, including Bacillus, Arthrobacter, Lactobacillus, Ilyobacter, Clostridium, and Lactococcus. Haria is a fermented rice beverage crafted using a starter culture (Ranu dabai) to boiled rice grains. It is a casual drink in daily life and is also utilized to alleviate ailments such as fever, headache, and nausea. Including herbal plants in the starter culture enhances the beverage, providing it with beneficial organoleptic and therapeutic properties. While extensive research has been conducted on Haria, we have not included Haria in our analysis. However, our study does encompass an examination of the plants used in the starter culture of Haria, an area where comprehensive research still needs to be done. The plant-derived phytocompounds demonstrated significant antioxidant activity, suggesting their capacity to reduce reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated during oxidative stress. Consuming the beverage may help overcome the growth of cancerous cells and slow the ageing process. Additionally, the herbal plants exhibit active antimicrobial properties, indicating a therapeutic role in inhibiting the growth of humanpathogenic bacteria and protecting the body from severe infections. "Eu" is a traditional mild alcoholic beverage made from millet and consumed by the Toto tribe in West Bengal. The preparation of Eu involves millet fermentation with the locally cultivated starter culture known as "Moaa." Ethnobotanical data and samples were collected through semi-structured interviews. Qualitative assays, such as acute toxicity, antioxidant analysis, gas chromatography-mass spectrometry (GC-MS), in vitro anti-inflammatory assay, molecular docking, and physiochemical assessments (pH and alcohol content), were conducted. The findings indicate that Eu has a low alcohol content (approximately 1-3%) and a high antioxidant capacity. GCMS analysis identified thirteen bioactive compounds. The starter culture employed in this brew comprises a diverse microflora. Identifying these microbes will enhance our comprehension of the brew and allow us to quantify its effects on the human system postconsumption. Leveraging advancements in Illumina sequencing technology, meta-profiling of the 16S rRNA (V3–V4 region) has become a valuable resource for scrutinizing the microbiome of various samples. We have utilized this tool to investigate the microbiome population of Eu. The microbial diversity found in the brew primarily consisted of probiotic and fermenting bacteria. Nutrient profiling of Eu assessed the levels of protein, carbohydrates, free amino acids, and free fatty acids. The nutritional content in the brew likely supports the growth of probiotic microflora. Reverse ecology-based network analysis unveiled substantial complementary interactions between humans and the microflora in the Eu. GC–MS analysis identified thirteen potent compounds in Eu, and an extensive network pharmacological study on these compounds revealed the anticancer properties of Eu, particularly against gastrointestinal malignancies. This was achieved by reducing the proliferation of malignant cells and forming pro-inflammatory cytokines. The probiotic microflora and the Eukaryotic Yeast also play a crucial role in fermentation. Several yeast cells were isolated from the starter culture, and from the isolated cultures based on biochemical screening, three strains were selected for identification. Molecular phylogenetic analysis based on ITS region (ITS1 and ITS4) sequencing of these three yeast isolates showing similarity with Saccharomyces cerevisiae. The MTT assay against the ACHN human renal adenocarcinoma cell line determined the cytotoxicity of Eu. The brew showed no cytotoxic effect. Whole metagenomic sequencing, or WMS, is particularly valuable in studying complex microbial ecosystems, where diverse microbial communities coexist. Whole metagenomic sequencing was done on the starter culture of Eu; the result showed .42% of Archaea, 60.80% of Bacteria, 22.84% of fungi, 11.95% of protozoa and 2.95% of Viruses within the sample. In vivo, acute and sub-acute oral administration toxicity study was conducted to show the efficacy of the drink Eu on a rat model. In the acute toxicity study, female and male Wistar rats received oral administrations of Eu of doses 250 and 500 mg/kg over 28 days following OECD Guidelines 425. In the sub-acute study, no significant reduction or gain of weight was observed when compared with the control group. The haematological and histopathological study also showed no significant alteration of the parameters when compared with the control. The spleen weight of both males and females was changed slightly compared to the standard, but this change is not dose-dependent. The result of sub-acute toxicity showed that an adequate amount of the drink.
Open Access
Selected underutilized fruits of Northeast India for overall health improvement of humankind
(University of North Bengal, 2023) Banerjee, Swarnendu; Sen, Arnab
The North-Eastern hill region in India is a significant agro-biodiversity hotspot with underutilized ethnomedicinal fruits essential for rural life. However, these fruits are often unknown due to lack of documentation, shrinking forest areas, and disappearance of indigenous culture. Plantderived products, particularly fruits, offer numerous benefits and are a promising alternative to current health -promoting medications due to their less expensive and less harmful side effects. Hence, the present study was conducted to collect wild edible fruits with ethnomedicinal values from India's North-Eastern hill region, considering their ethnomedicinal importance. Since these fruits are ethnomedicinal and nutritionally essential, this present investigation aims to explore bioactive phytocompounds from underutilized fruits and their health-beneficial role against some diseases. Fourteen fruit samples were collected from different locations in three states (West Bengal, Meghalaya, and Tripura) among them four fruits: Elaeagnus pyriformis (EP), Baccurea ramiflora (BR), Phyllanthus acidus (PA), and Prunus nepalensis (PN) were selected for further experiments while the remaining fruits were not considered based on DPPH assay. These four fruit extracts [EP (80.91±0.264%), BR (78.864±0.11%), PA (78.053±0.78%), PN (81.56±0.57%) at 200 μg/ml] exhibited higher free radical scavenging activity than the others when compared to the respective standard (ascorbic acid). With increasing concentrations of extracts (50-200 μg/ml), these fruit samples showed promising reactive oxygen species (ROS) scavenging power in the case of other in vitro assays [hydroxyl radical, hydrogen peroxide, nitric oxide, and hypochlorous and total antioxidant assay]. The study showed a strong positive correlation between antioxidant scavenging assay and total phenol and flavonoid content for all four fruit extracts, where EP showed the highest phenol (73.74631 ± 1.171 mg GAE/g DW) and flavonoid content (39.3726±0.6661 mg QE/g DW). High phenol and flavonoid content have been proved by FTIR analysis where EP, BR, PA, and PN extracts indicated the presence of significant peaks for polyphenolic OH, alkane, carboxylic acid, and amide-I at around (3419- 3439 cm-1), (2920-2927 cm-1), (1732- 1740 cm-1) and (1625-1648 cm-1) respectively. The present study was extended to analyze the identification of active phytocompounds in EP, BR, PA, and PN extracts using the GC-MS method. A total number of 38 phytocompounds have been identified in EP (11 compounds), BR (8 compounds), PA (8 compounds) and PN (11 compounds). It has been reported that fatty acids like octadecanoic acid (stearic acid), dodecanoic acid (lauric acid), and hexadecanoic acid (palmitic acid) were present in the fruit extracts that humans need in their diets. Apart from these, ellagic acid, Ƴ-sitosterol, 1,2- Longidione, n-hexadecanoic acid, and octadecanoic acid, 4H-Pyran-4-one, 2,3-dihydro-3,5 -dihydroxy-6-methyl each have anticancer, antidiabetic, nephroprotective, hypocholesterolemic, antibacterial, and antiinflammatory properties. Therefore, based on the study above, it is possible to indicate that EP, BR, PA, and PN extracts can potentially become powerful antioxidative agents in the future. Further LC-MS analysis allowed the identification of more than 75 polyphenols, including flavanols, flavonoids, anthocyanidins, alkaloids, steroids, vitamins, triterpenoids, phenylpropanoids, phenolic acids, and derivatives from four fruit samples (EP, BR, PA, PN). It has been reported that vitamin B9 has antioxidant and neuroprotective properties among these bio compounds and reduces the chances of lipid peroxidation. Reports suggested that flavonoids (trifolin, quercetin-3β-D-glucoside, kaempferol), flavonols (rutin), anthocyanidins (malvidin), phenolic acids (quinic acid, gallic acid, phenolic acid), phenylpropanoids (rosmerinic acid) are potent antioxidant and ROS scavengers. These have high antioxidant, anticancer, anti-inflammatory, hypolipidemic, and immunomodulatory properties. Fruit extracts were investigated against two Gram-positive (Bacillus subtilis, Staphylococcus aureus) and two Gram-negative (Escherichia coli, Salmonella typhimurium) pathogenic bacterial strains to see antimicrobial activity. Among all fruit extracts, PA showed perfect growth inhibitory activity against gram-positive and gram-negative bacteria at low doses (5mg/ml). At high doses (10mg/ml), EP, BR, PA, and PN (all four fruit extracts) showed a significant zone of inhibition against S. typhi (11.576 mm), E coli (14.256 mm), B. subtilis (13.964 mm), S. aureus (15.382 mm) respectively. The study demonstrated that active phytocompounds (beta-sitosterol, octadecanoic acid, γ-sitosterol, and squalene from fruits like EP, BR, PA, and PN, respectively) have potential growth inhibitory activities against pathogenic strains, which suggests these fruits could be used as herbal remedies for microorganism-related disorders. When the ACHN human renal adenocarcinoma cell line was exposed to varying doses of four fruit extracts (EP, BR, PA, and PN), after 48 hours of treatment, the MTT test results offered an interesting concentrationdependent decrease in cell viability, which ranges between 45% to 49% at the highest dose (300 μg/ml) of four (EP, BR, PA, and PN) fruit extracts. This MTT assay data was also supv ported by in silico molecular docking analysis where selected phytocompounds (Malvidin, Rosmerinic acid, Ellagic acid Quercetin for EP, BR, PA, and PN respectively) showed potential inhibitory activities against the MDM2 protein, which is a positive regulator for adenocarcinoma. Probably, this was the first report on the cytotoxic effects of these underutilized fruit extracts on the ACHN cell line. An increasing dose ((5, 10, 20, 40 mg/ L) of resveratrol (a potent polyphenol from EP) demonstrated protection against H2O2-induced oxidative injury and increased sperm viability by upregulating several frontline antioxidant and non-antioxidant enzymes such as SOD (165-172%), GSH (18.5- 91.43%) respectively, reducing ROS level [NOX (39-55%)] improving altered lipid metabolism and histological architectures (Sertoli cells and seminiferous tubules regenerates). In silico molecular docking study also supports this data where resveratrol showed good inhibitory potential (- 7.8kcal/mol) against stress protein NOX2. The different resveratrol treatments had better antioxidant activities than the standard antioxidant, ascorbic acid (AA). Therefore, this phyto compound might be a promising medicine against stress-mediated male infertility problems. Fruit wines (fermented alcoholic beverages) are traditionally popular in the cool climatic zones of Northeast India. A study on fruit wines in Northeast India found high antioxidant activity, medicinal potential, and significant anti-cancer activity in breast cancer cell-line studies. Metagenomic analysis revealed diverse probiotic bacterial species in the wine sample's microbial population, playing an essential role in anti-cancer properties. Overall, the study suggests wine has therapeutic and health benefits. The study also describes the green synthresis of silver nanoparticles (AgNPs) from silver nitrate utilizing EP fruit juice in an optimized process, which caused the solution's color to shift from yellow to brownish. UVVis spectroscopy, SEM, FESEM, EDX, XRD, and FTIR were used to characterize the AgNPs thoroughly to determine the existence and degree of bioactive compounds as a capping and reducing agent. Additionally, the antibacterial activity of AgNPs against human pathogenic microorganisms was shown to be substantial, and their antioxidant effectiveness was assessed. The anti-apoptotic protein Bcl- XL was employed in an in-silico molecular docking investigation to determine the efficacy of AgNPs as a powerful anti-cancerous agent. According to the results, nanoparticles are deemed harmless as they can alter how cancer cells typically initiate cell death by blocking their ABC transporters. A similar study was done using BR fruit juice, and surface morphology, size, and stability of biogenically synthesized AgNPs were determined by SEM, FESEM, HRTEM, EDX, XRD, and DLS. In this study, DPPH and other in vitro antioxidant assays explored the effect of these nanoparticles in treating oxidative stressrelated disorders. The antimicrobial assay revealed that AgNPs have potent antibacterial activity against both above mentioned gram-positive and gram-negative bacteria. Two human breast cancer cell lines were used in an MTT test to determine the anticancer effects. Results indicated that AgNPs were cytotoxic to the cell lines in a dose-dependent manner, with IC50 values of 110 mg/ml and 140 mg/ml, respectively, after 48 hours of treatment. We may conclude from this work that AgNPs are highly effective in preventing cancer. The study synthesized nanoparticles from Phyllanthus acidus fruit juice to investigate its clinical effects. The study compared the efficacy of raw fruit juice and nanoparticles on gentamicin-induced nephrotoxicity in a mouse model. The present study administered gentamicin (negative control) intraperitoneally to induce nephrotoxicity in the mice model. Several in vivo antioxidant and biochemical tests were performed to measure the efficacy of low and high doses of fruit juice and juicesynthesized nanoparticles in preventing renal injury. Results revealed that creatinine and urea levels significantly deteriorated by up to 1.13% and 1.49% in the high-dose P. acidus nanoparticles (PANH) group compared to the negative control group. After the application of PANH, major antioxidant (SOD, CAT) and nonantioxidant (GSH) enzymes were significantly enhanced compared to the gentamicin-treated group. Histopathological examination revealed that the glomerulus structure reappeared significantly in the PANH group. In silico molecular docking, analysis showed an inhibitory effect of fruit juice and fruit juice synthesized nanoparticles against inflammatory protein NFƙb. Findings indicated that PANH was more ameliorative over a high dose of P. acidus fruit juice (PAJH) towards kidney injury. Our findings generate scopes to explore the therapeutic potentials of PANH in treating kidney disorders. Now a days arsenic, a toxic metalloid, poses a significant threat to human health and agriculture. In India, the highly nutritious Vigna mungo (L.) Hepper (Blackgram) pulse crop, Urad, is highly contaminated, leading to failure in germination and reduced crop yield. This contamination is particularly prevalent in West Bengal, Murshidabad, and Malda districts, where blackgram is cultivated alongside other cash crops. The build-up of arsenic in grains endangers agricultural productivity and food security because arsenic stress inhibits seed germination and plant growth. To reduce Asphytotoxicity in plants and seedlings before they reach the reproductive stage, this study investigated the viaviii bility of seed-priming black gram plants with biogenic zinc oxide nanoparticles (ZnONPs). ZnONPs under stress considerably increased the germination rate of blackgram seeds (20– 23.33%) and other morphophysiological characteristics. Adding ZnONPs reduced the accumulation of malondialdehyde (21–69%) and reactive oxygen species (ROS) (hydrogen peroxide) by 23–67%. ZnONPs also led to an increase in the activity of antioxidant enzymes (superoxide dismutase by 28–68%, catalase by 20– 42%, guaiacol peroxidase by 19–49%, and ascorbate peroxidase by 26–39%) and the accumulation of osmoregulators. The findings suggest that ZnONPs significantly reduce Astoxicity by preventing its transfer from the root to the shoot. This work provides the first proof that ZnONP-based nano-priming effectively reduces Asstress in blackgram, indicating the possible application of this technique as a stress-relieving agent in Ascontaminated regions to boost yield and growth. Another important legume in the food chain such as peas (Pisum sativum L.) are rich in nutrients. However, arsenic (As) in the agroecosystem provides a considerable bottleneck to its productivity because of its severe effects on ontogeny, particularly in Southeast Asia. This work proposes biogenic ZnONPs as a rhizospheric nanoremediation technique to avoid Asgenotoxicity and increase crop production. In the negative control, germination rate, shoot length, and root length dropped by 62%, 16%, and 14.9%, respectively, above the normal control. As-toxicity caused fast oxidative bursts with worsening morphophysiological properties. Negative control experienced membrane damage (82.75% increase) and electrolyte leakage (2.6-fold increase) as a result of ROS buildup (12.8 and 9-fold increase in leaves and roots), overworked antioxidant defense, and loss ix of cellular homeostasis. According to the study, root tip cells under stress significantly increase nuclear area, nuclear fragmentation, and micronuclei formation. These findings suggest severe genomic instability and increased programmed cell death (with a 3.3-fold increase in early apoptotic cells) due to leaky plasma membranes and unrepaired DNA damage. ZnONPs adsorb into the rhizosphere and reduce As-toxicity, they improve the antioxidant response and reduce As-uptake in peas. As-interference is reduced by increased vacuolar sequestration of As due to improved phytochelatin fabrication. Better photosynthetic activity (1.3–1.9-fold increased chlorophyll autofluorescence), increased pollen viability, and less genotoxicity (reduced tail DNA in comet assay) were observed. The flowering time was also better (7.74– 19.36% reduction in flowering delay) with greater transcript abundance of GIGANTIA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) genes. Pod number and seed zinc concentration increased up to a maximum of 37.5% (1.67-fold), whereas seed arsenic content was reduced when treated with ZnONPs. In our experiment, the maximum ZnONPs dose (400 mg/L) caused NP-toxicity in pea plants; however, up to 300 mg/L, the best stress alleviation was seen. Although blackgram and pea are highly significant pulse crops, the use of nanoparticles in reducing heavy metals or metalloid stress in these crops has never been reported. Through this investigation, we hope to fully assess the viability of using ZnONPs to reduce stress caused by As. Therefore the detailed study on underutilized fruits have shown their potentiality against several diseases like cancer, infertility, nephrotoxicity and improves human health in a drastic way.
Open Access
Evaluation of silica nanoparticles and their functionalization in the alleviation of salinity stress in two legumes -Lens Culinaris and Glycine Max
(University of North Bengal, 2023) Sarkar, Mahima Misti; Roy, Swarnendu
Soil salinity is one of the major environmental stresses that imparts drastic effects on plant growth and development. The adverse effects of salinity affect the overall plant health, resulting in impaired physiological and biochemical attributes. Poor health ultimately causes a loss in plant production rate and nutritional values. To overcome the negative effects of salinity and to improve the yield of crop plants, major emphasis has been given to the application of several fertilizers, pesticides, and fungicides. Moreover, the development of genetically modified crops has been advocated for the development of salt-tolerant crop plants. However, these have some drawbacks, such as genetic modification being timeconsuming and cost-consuming and applying chemical fertilizers hampering soil fertility. Moreover, the efficiency of fertilizers, considered important for improving crop yield, reduces soil fertility due to over-application, resulting in residual toxicity on crop plants. In this context, in the last decade, nanotechnology has emerged as an alternative strategy for the betterment of plant health as well as environmental stress alleviation. The idea behind using nanoparticles in crop improvement was to improve the efficiency of the bulk materials already used as agrochemicals. Because of having a high surface area to volume ratio, the nanomaterials facilitate easy uptake and render them more interactive with the cellular active components, thereby amplifying their efficiency. Though silica is not an essential element in plants, its deficiency creates various problems; thus, it is considered a semi-essential element. The application of silica nanoparticles (SiNPs) has excellent potential for crop improvement because it increases silica availability to plants. The synthesis of any nanoparticles demands an optimization process through which we can understand the ratios of components that should be used to get the actual shape, size, and surface charge. In this purview, the second chapter presents the synthesis, optimization, and characterization of SiNPs. SiNPs were optimized by varying the three important ingredients (TEOS, ethanol, and ammonia) through ‘Design Expert 13’ based ‘mixture design’, which revealed that the size of the SiNPs can be modified but not the surface charge. Using mixture design, we successfully obtained ~50 nm-sized SiNPs, which was confirmed through several analytical techniques. To study the potential of SiNPs in salinity stress alleviation, four concentrations of NaCl (for seedling and vegetative stage – 0, 100, 200, and 300 mM; for reproductive stage – 0, 200, 400, and 600 mM) in combination with four concentrations of SiNPs (0, 1, 5, and 10 g/L) were applied in two legume crops – lentil, and soybean, respectively (Chapters 3 and 4). The results depicted that both plants had better germination attributes under salinity after the application of SiNPs. In the seedling and vegetative stages, SiNPs were found to improve the plant’s health under salinity through improved plant height, relative water content, photosynthetic pigments, ionic and osmotic balance, antioxidant defense (enzymatic and/ non-enzymatic), membrane stability and reduced ROS accumulation. Similarly, in the reproductive stage of both legumes, applying SiNPs was also observed to reduce the salinity stress impacts through improved plant health, yield, and nutritional aspects of the seeds. The use of bare SiNPs has already been proven to have beneficial attributes for crop plants under salinity. However, very little research has focused on applying functionalized nanoparticles for crop improvement under environmental stresses. Surface functionalization of these nanoparticles with bioactive molecules has the potential to increase their efficiency in this regard. Thus, in chapters 5 and 6, the SiNPs surface was functionalized with two important sugars/osmolytes – glucose (GSiNPs) and trehalose (TSiNPs), respectively. Both surface functionalizations were confirmed using several analytical techniques, including FTIR, XRD, DLS with zeta potential, UV-visible spectroscopy, SEM, EDS, TEM, and AFM. Applying both GSiNPs and TSiNPs improved the health of lentil and soybean seedlings through improved height, photosynthetic pigments, ion balance, and antioxidant defense under salinity stress. In both plants, the improvement was found to be more profound with the use of GSiNPs and TSiNPs compared to the bare SiNPs. The bioassay experiments also proved that the functionalization of the SiNPs surface with glucose/trehalose increased the bioavailability and uptake of both silica and sugars in the seedlings. Thus, these studies can provide ample evidence of the greater efficacy of surface-functionalized nanoparticles in palliating salinity stress in plants. Apart from the morphological, physiological, and biochemical analyses, we performed the molecular and genetic background of the beneficial effects of surface functionalized SiNPs (TSiNPs) by performing the whole transcriptome analysis (RNA-Seq). The results revealed that the TSiNPs have regulatory roles on various genetic expression, which modulates various protein expressions, pathway functions, cellular components, molecular functions, and biological processes to confer progressive health benefits for lentil seedlings under salinity. More specifically, some of the genes involved in the providence of salinity stress tolerance (Sodium hydrogen exchanger, Potassium transporter, Cytochrome b6, sugar transporter, and ABC transporter G family member) were expressed highly in the presence of TSiNPs. Along with their beneficial effects on crop improvement, various nanoparticles have also been found to have toxic effects on plants, animals, and environmental elements. So before proceeding to the filed application and commercialization, the SiNPs must also be checked for their toxicity attributes. In this purview, the cytotoxicity and genotoxicity of the synthesized SiNPs were evaluated on the Allium cepa root tip, considering their applied concentration and size. Further, the toxicity effects of SiNPs were attempted for attenuation using surface functionalization with sugar molecules – GSiNPs and TSiNPs. The results showed that the SiNPs showed toxic effects after the 100 g/L concentration, and the 30 nm and 100 nm sized SiNPs showed more toxic effects than the 50 nm-sized nanoparticles. Further, the GSiNPs and TSiNPs were found to reduce the toxicity level of SiNPs even at higher concentrations (> 100 g/L). A toxicity study was also performed on soil microflora, and the results showed that the SiNPs, GSiNPs, and TSiNPs improved the soil microbial communities (mixed soil bacteria, phosphate solubilizers, nitrogen fixers, and silica solubilizers) in control (0 mM NaCl) as well as 300 mM NaCl stress conditions. Finally, the SiNPs, GSiNPs and TSiNPs were developed into nanoformulations (slowrelease type) using sodium alginate as internalizing polymers. The results of Chapter 9 revealed that the alginate internalization makes the silica and/or sugar release from SiNPs, GSiNPs, and TSiNPs at a slower rate, considering the effect of incubation time, pH, and temperature. These nanoformulations also improved the seedling growth even better than the bare and functionalized SiNPs, even in the presence of NaCl stress. However, the cost of production at the laboratory scale is relatively high, and our future emphasis is to make these nanoformulations cheaper so that they can handed over to the farmers for field application.
Open Access
Taxonomic and phytogeographical studies on the flora of Mirik and its environs in Darjeeling district
(University of North Bengal, 1987) Mukhopadhyay, Chittaranjan; Basu, P. K.; Ghosh, R. B.
In recent years much attention has been given to Mirik to establish it a beautiful hill resort in connection with the development of tourism in the region. It is situated at an altitude of 170Dm with its own special charm, located at 49 km from Darjeeling and 52 km .from Siliguri. This hill resort has a special attraction for having a magnificent lake fed by perennial streams. It is under the Mirik P.S.of Darjeeling district in West Bengal and is located between 26°47'N. lat. and 26°55'N. lat., 88°8'E. long. and 88°15'E. long. It claims its special identity in respect of its strategic position in the Sub-Himalayan hilly tract in the northern region of West Bengal. Its flora is significant from the scientific, cultural and utilitarian view points. It has a wide range of habitats which provide ecological diversity. Phytogeographicall y also it occupies a key position. The economy of Mirik and its environs is primarily based on agriculture, forestry and plantation. Like all other parts of the world, the ecosystem of the region has also been observed to be the most gravely threatened now-a-days. Due to large sc.ale destruction without adequate replacement of forest trees, forest wealth has been diminished and due to Jack of plant cover, soil is subjected to the combined influence of erosion, leaching, insolation and radiation. Thereby, much of the normal biogeochemical cyde is. lost causing Jack of regeneration of forest plants. Recently, due to policy at the National level, large scale plantation is being encouraged in connection with Eco-Development Programme. But the proper management of the forest in a region is possible only with the utilization of the local plants in the area. It is, therefore, of primary importance to understand the. present day plant resources of the region. As lhe development and distribution of floristic elements are very much dependent on the ecological condition of a particular region, emphasis has been given to study the floristic composition, ecological conditi'on of the . re_gion with special ~tress on the behaviour of different plant species from phytogeographical point of view. During investigation some ecological observations on Mirik and its environs have been made after accumulation of information from various sources. In this connection collaboration has been made with the National Atlas and Thematic Mapping Organisation, Government of India. Information so far represented in connection with Mirik P. S. may be considered for the first time to report in this .field of work. During the preparation of / topographic section, it has been noted that less than 700m elevation shows the- lower erosional surface, 700-1000m the middle erosional surface and above 1 DOOm represents upper erosional surface of the mountains. Mirik itself lies in the. highest part of the region in Mirik P.S. The height gradually slopes down towards south-east havi~g average height of 300-600m above sea level. The surface configuration of this area undoubtedly presents a complex physical environment due to. different Geomorphic processes. The study on drainage and the river system of the region has. specially been emphasized due to the fact that now-a-days catchment area has been noted to be a very important from ecological point of view. In co-nnection with the study on this aspect, it has beeri noted that the Mechi and the Balason are the two main rivers with a large number of tributaries and branches, the courses of which have been taken into consideration during the preparation of map. The central part of the region acts as main water divide line. The water divide area is having a slope of less than 20% as calculated from the relief and the slope is maximum where lhe land is merging with the boundary rivers. Thus along both the rivers, the Mechi and the Balason, the slope increases to more than 50%. Five different zones of soil have been prepared for Mirik P.S. These are Red Podosol, distributed in the northern part of the region, Brown Earth, represent)ng the major part of the area, Ash Grey Soil with association of Red Soil, representing a narrow-strip towards the southern part, Whitish Grey Soil adjacent to the previous one in southern direction and Grey Soil with association of yellowish brown Soil representing the extreme southern part of the police station. Out of the study on the earlier records on climatic factors, it has been noted that since 1 Q50 the rainfall has been gradually declining from 3477.51 mm (1850-1855) to 2560.00 mm (1982-1987). Total rainfall in the year of 1988 has been observed to be 2039.4 mm. Thus the vegetation, now-a-days, is beinc:J subjected to stressed condition as compared to earlier days. Due to rapid growth of urbanisation, the growth of human population has been observed to be at increased state and for this reason biotic influence on vegetation is inevitable. This "increase in population coupled with the increased demand on natural forest areas for cultivation of cereals for food and for fuel, forest areas are being destroyed at a greater pace, year after year. Similarly, increased communication even with remote villages facilitates more movement of people from outside along with their domestic animals and consequently more interference with the vegetation. The major part of this area has been noted to have population density of 1-3 persons/ha. The small area of the central part has more than 9 persons/ha, and a small area in the north has.been observed to cover less than 1 person/ha. A very significant feature observed here in the study of landuse is the extensive existence af tea gardens. Crop land occupies a very little area which is distributed mostly in the northern part of the region. A map has been prepared to represent rural settlements, forests, scrubs and wastelands. · The primary or vergin forest has been noted to become restricted in the region. Majority of forest patches are secondary in nature. Vegetation of this area has been classified on the basis of physiognomy and structural features of the plant in relation to "the altitude of the hill and climatic conditions, specially the rainfall. Four vegetational zones i.e. Tropical Deciduous Sal Forests (Plantations; 1 00-500m), Tropical Evergreen (500-1200m), Subtropical Evergreen (1200-1800m) and Temperate Mixed Zone (1800-2630m) have been marked out and different plant species in various zones of the primary forests have been worked out. A change of vegetational structure for secondary forest formation due to the influence of various factors have been noted. Different species available in different zones of primary and secondary forests have been enumerated. Water reservoirs in different zones of Tropical, Subtropical and Temperate regions have been observed though typical aquatic vegetation is poorly developed, the marshland vegetation has been studied in details. Cultivated and other useful plants with special reference to food, medicinal and ornamental value available in the region have also been recorded. Special emphasis has been given on the survey of various plant materials used by the local tribes/hill people. Besides, common trees yielding timber, fibre and fruits have been identified. In connection with the taxonomic study on the flora of Mirik and· its environs in Darjeeling district, much emphasis •has been laid upon the collection of plants, preparation of herbarium sheets and identification of them after ·consultation with different herbarium sections available. Nomenclatu' re and citation, short description, flowering and fruiting periods, local name, if any, notes on the frequency of distribution, plant association, economic use, if any, of 1059 taxa including 71 cultigens of Angiosperms and G~n;nosperms have been taken into consideration. Out of which again 987 and 67 species of Angiosperms are represented as wild ~nd cultigens respectively. Gymnospermous taxa have been found to be represented by Cryptomeria japonica (L. f.) D. Don in wild state and 4 other cultigens. The system of Bentham and Hooker (1862-1883) with. some modifications based on recent knowledge has been· followed during the representation of the sequence of families in the flora. Monocots include 300 species (excluding the cultivated 11 species) unde·r 136 genera and. 14 families. Similarly, Dicots cover 687 species (excluding the cultivated 56 species) under 350 genera and 95 families. "Based on total number of collection (excluding cultigens), percentage occ:urrence of family, genera and species of Dicots have been observed to be 86.36%, 71.87% and .69.53% respectively; Similarly, in Mof\OCots, family, genera and species have been represented by 12.73%, 27.93% and 30.36% respectively. The approximate ratios between the family, genus and species of the Angiosperms have been worked· out. The Monocot-Dicot ratios in terms of family, genus and species have been calculated to be 1:6.8, 1:2.6 and 1:2.3 respectively. The genus-species ratio for the Angiosperms is 1:2 approximately. Twenty ·different families in order of dominance have been worked out on the basis of the number of taxa incorporated in each family. Orchidaceae has been noted to include maximum of 131 taxa under 41 genera. On the other hand, each of Apiaceae and Acanthaceae includes minimum of 13 ta?
Open Access
Studies on genetic variability and distribution of begomoviruses affecting economically important crops of sub-himalayan plains of North-East India
(University of North Bengal, 2023) Karmakar, Arup; Biswas, Kajal Kumar
Diseases caused by viruses are responsible for poor crop yield in several vegetable crops. Several million rupees are lost worldwide due to infection of our crops by viruses. Begomoviruses are the most devastating plant pathogens that causes high amount of crop loss every year in India. Several vegetable crops are cultivated in sub-Himalayan West Bengal and Assam. Due to yearlong conducive atmosphere production of the vegetable crops are also popular to the local farmers. The present work entitled “Studies on genetic variability and distribution of begomoviruses affecting economically important crops of Sub- Himalayan plains of North-east India” consists of the following objectives such as i) Identification of begomoviruses infecting economically important crops of sub-Himalayan plains of North-east India using Polymerase chain reaction (PCR) and Rolling circle amplification. ii) Sequence analysis and determination of variability among begomoviruses and their strains using bioinformatics tools. iii) Eco-friendly management of important Begomovirus diseases using botanicals. To know the begomoviral diseases of the present study area, a survey was carried. Around 20-75% begomoviral disease incidences have been found in some vegetable crops. Ten important crops of the present study area are tomato, cucumber, potato, kenaf, pointed gourd, papaya, loofah, pumpkin, chilli and ladies finger. Begomoviral symptoms were found in all the ten crops, resulting to significant crop loss. Major symptoms observed were mild to severe yellow mosaic, upward leaf curling, leaf rolling, stunted growth, leaf deformation, networking of yellow veins, followed by thickening of veins and vein lets, puckering, stunting of whole plant and reduced fruit yield. Altogether 55 samples were tested through PCR using different primer sets. Among the 55 samples, 17 samples were positive for begomoviruses. Out of the 17 samples eight were from tomato, seven were from cucumber; one was from kenaf and one was from pumpkin plants. Out of the 16 Begomovirus isolates of the present study, nine were identified as Tomato leaf curl New Delhi virus (ToLCNDV), two were identified as Radish leaf curl virus (RaLCV), two were identified as Papaya leaf curl virus (PaLCuV), one was identified as Tomato leaf curl Karnataka virus (ToLCKV), one was identified as Tobacco curly shoot virus (TbCSV) and one was identified as Ageratum enation virus (AEV). Among the ToLCNDV isolates, three were detected in tomato (Accssion Nos. KX108860, KX817297and MZ516898), five in cucumber (Accssion Nos. KY783746, KY807530, MG721011, MG721012 and MG721013) and one in pumpkin (Accssion No. MG721010). Two isolates of tomato of the present study were found to be Radish leaf curl virus [Accession no. MK333456] as they showed 97% sequence similarity with other RaLCV sequences previously submitted in GenBank. Based on 94-96% sequence similarities following results were found: i) Papaya leaf curl virus detected from tomato (Accession No.MZ516896) and cucumber plants (Accession No. MK333457). ii) Tobacco curly shoot virus was also detected from cucumber (Accession No. MG721014) plants iii) one tomato sample was found to contain Tomato leaf curl Karnataka virus (Accession No. KX108859) iv) One Begomovirus species Ageratum enation virus [Accession no. MZ516897] infecting tomato was identified. That AEV showed 95% sequence similarity with the other AEV of the GenBank. In this study, infected leaves of selected plants like tomato, cucumber and kenaf were used for Begomovirus detection through Rolling Circle Amplification (RCA). High molecular weight DNA obtained by RCA of the infected sample was visible in 1% agarose gel under UV-transilluminator, indicating the amplification of circular DNA. RCA product (5 μl) was digested with a number of different restriction enzymes to select an enzyme with a single restriction site in the DNA components. XbaI and BamHI were found to have a single site in both DNA-A and DNA-B component. In the HindIII digested sample two bands of 2.7kb and 1.4 kb were found. In the Pst digested product, three bands of 2.74kb, 1.4kb and 1.45kb were found. In the EcoRI digested sample, two bands of 2.74 kb and 1.4kb were found. The band 2.7kb probably indicate the presence of whole genome of Begomovirus either DNA- A or DNA-B, and the band of about 1.45 kb probably indicate the presence of beta-satellite or alpha-satellite until sequenced. The amplified fragment of 2.74kb were purified and ligated in to pGEM-3Z vector which was also digested by the same enzyme XbaI and cloned according to manufactures instruction. After transformation in to DH5α strain of E.coli, white colonies were screened for the gene of interest by PCR and restriction digestion with XbaI. Positive PCR white colony was selected for sequencing. Three isolates (To/Guw, Cu/Khr and K/Blg) were found positive following RCA and RFLP. Those three isolates produced distinct bands of ~2.7kb and ~1.4kb. The RCA product was subjected to PCR by β satellite specific primer and the amplicons were found to the betasatellite (after cloning and sequencing). Out of the two positive samples, one was identified as Tomato leaf curl betasatellite (ToLCB) infecting tomato (Accession No. MK333455) as it showed 96% sequence similarity with ToLCB. Another one was identified as Cotton leaf curl Multan betasatellite (CLCuMB) infecting Kenaf plants (Accession No. MK358823), which showed 98% sequence similarity with CLCuMB upon BLASTn analysis. The recombination analysis by RDP4 showed that the isolate GUW-01 (Tomato leaf curl betasatellite; Accession No. MK333455) was a recombinant having major parent Tomato yellow leaf curl Thailand betasatellite (TYLCTHB, Accession No.GU058327), and minor parent TYLCTHB (Accession No. GU058324) infecting tomato in China. This suggests that the ToLCB isolate GUW-01 (Accession No. MK333455) may have derived from recombination and it contains sequences derived from TYLCTHB (Accession No. GU058324). The sequences of AEV infecting tomato were clustered together with other tomato infecting AEV isolates in the phylogenetic analysis. PaLCV isolates of the present study clustered with PaLCV infecting Carica papaya whereas PaLCV from other hosts showed separate clusters. RaLCV isolates infecting tomato of the present study showed close relationship among them and clustered with tobacco infecting RaLCV isolate. Phylogenetic analysis of TbCSV isolate infecting cucumber showed that the isolate formed separate small cluster with Phaseolus vulgaris infecting TbCSV whereas the isolates infecting other host formed different cluster. Tomato infecting ToLCKV of the present study clustered with tomato infecting ToLCKV of GenBank and showed close relationship with tomato infecting isolates found worldwide. Phylogenetic analysis of the present nine ToLCNDV sequences along with other ToLCNDV sequences obtained from public database revealed close relationship among them and clustered together. Pumpkin-infecting isolate of the present study showed close relationship with other pumpkin-infecting ToLCNDV isolates. Cucumberinfecting isolates was also clustered together with other cucumber-infecting ToLCNDV isolates except one isolate which positioned alone. But tomato infecting isolates clustered with cucumber-infecting In phylogenetic analysis Cotton leaf curl Multan betasatellite (CLCuMuB) isolate showed close relationship and clustered together with other CLCuMuB isolates whereas Tomato leaf curl betasatellite (ToLCB) isolate showed close relationship with Tobacco curly shoot betasatellite (Accession No. KX857135) and also clustered together with that isolate. In the present study, four enzymes (peroxidise, β-1,3-glucanase, Polyphenol oxidase and PAL) activity was studied following application of the chemical inducers. BABA treated and BABA treated-inoculated plants showed highest peroxidase activities followed by AABA treated and AABA treated-inoculated plants. Highest increase in β-1,3-glucanase activity was found after 12 days of treatment in the GABA treated-ToLCNDV inoculated plants followed by BTH treated-ToLCNDV inoculated plants as compared to the control plants. Polyphenol oxidase activity was also increased in all treated plants except BABA treated plants. Among the inducer treated plants, BABA treated plants showed maximum PAL activity. GABA and BTH treated plants showed comparatively less PAL activity. Among the „plant extract-treated‟ plants, P. betel treated plants showed Maximum peroxidase enzyme activity followed by A. indica treated plants. Treated-inoculated plants also showed significant increase of enzyme activity. Among the experimental plants, A. indica leaf extracts treatedinoculated plants showed highest enzyme activity followed by C. sinensis treated-inoculated plants. Botanical plant extracts also increased the level of β-1,3-glucanase activity in treated plants. Among the treated and treatedinoculated plants, C. sinensis treated and treated-inoculated plants showed maximum enzyme activity followed by P. betel treated and treated-inoculated plants. PAL enzyme activity was also studied after exogenous application of the plant leaf extracts. Here in this study A. indica leaf extract treated and treated-inoculated plants showed highest enzyme activity than the control. Piper betel treated-inoculated plants also showed an increased level of PAL activity. PPO activity was also significantly increased in the plant extract treated and treated-inoculated experimental plants of this study. B. diffusa treated and treated-inoculated plants showed highest enzyme activity followed by P. betel treated and treated-inoculated plants. Some of the new findings are (i) New host (tomato) infected by Radish leaf curl virus, (ii) New host (Cucumber) infected by Papaya leaf curl virus and Tobacco curly shoot virus (iii) Tomato leaf curl New Delhi virus infecting cucumber is being reported for the first time from this region and (iv) betasatellites of begomovirus isolate like Tomato leaf curl betasatellite infecting tomato plants along with their recombination events. Genetic variability of the isolates was also studied. Finally management of the begomoviral disease caused by Tomato leaf curl New Delhi virus has been done by four chemical inducers and five botanical leaf extracts. Some of the chemical and botanical inducers showed significant disease reduction.
Open Access
Cytogenetic study of the ethnic population of North Bengal, with special reference to constitutive heterochromatin polymorphism
(University of North Bengal, 1984) Agarwala, Rudra N.; Dutta, K. B.
Open Access
Seedling Invigoration of Mung Bean Sprouts Through Matrix Bound Selected Elicitors Including Nano-Chitosan Under Salinity Stress
(University of North Bengal, 2023) Sen Sujoy Kumar; Bhandari Jnan Bikash
The short-duration leguminous warm-season crop, Vigna radiata (L.) R. Wilczek is now grown globally in a balanced manner, particularly in emerging nations. It maintains a special place in the human diet as a sprout, dahl, and in numerous other forms due to its wide nutritional range, speedy and easy preparation, excellent digestion, and anti-flatulent characteristics. Due to its high amounts of folate and iron and excellent protein content, mung bean is in high demand and commands a premium price, which makes the farmers happy. The lack of nutrient-dense food, specifically for poor vegetarians, combined with expanding population associated with increasing global food crises, and limited natural resources, made this nutritious food crop more crucial. Additionally, it has a symbiotic relationship with Rhizobium, which improves soil fertility by fixing atmospheric nitrogen and making it ideal for rice-based cropping systems and intercropping with other crops. Mung bean plants are occasionally broken up and buried in the soil to improve the nitrogen content of the soil. As a result, they also aid succeeding crop plants in meeting their own nitrogen needs. Despite having so many advantages, mung beans have received less attention than other pulse crops. Moreover, being a self pollinated pulse having a small sized genome, it might serve as a study model for other legumes. But, the problem of rising salinity, one of the most serious global environmental issues, has been adversely hampering the overall mung bean productivity world-wide. An estimated 1.5 million hectares of agricultural area are lost to production annually due to high salinity levels present in the soil. More than 45 million hectares of the irrigated area have been ruined by salt globally. Alarmingly, it is anticipated that salinity will deteriorate over the next few decades. Plants are negatively impacted by salinity stress in many ways, including water stress, ion toxicity, nutritional issues, increased lipid peroxidation, metabolic process change, membrane disruption, reduced cell division, etc. These negative consequences altogether retard plant growth badly affecting its ultimate survivality. To provide humanity with a sufficient food supply and a balanced diet, scientists and researchers should show more interest in this subject. As a result, it's crucial to find and create mung bean cultivars with sustained resistance to this negative environmental pressure. Numerous tactics were established over time to increase the quantity and quality of seedlings of various crop species. Seed priming is such a feasible, affordable, and successful method of reviving seeds. New priming techniques are constantly being developed around the world to improve the nutraceutical properties and yield of pulse crops. One such novel and innovative approach is Solid Matrix Priming (SMP). According to the literature review, SMP was relatively understudied compared to the other priming approaches and is being employed for the first time in mung bean (except our published papers). Impact of this less commonly used novel priming technique (SMP) in mung bean also demonstrates vast potential in seedling invigoration at a commercial scale that has yet to be investigated. Additionally, it is, in many ways, cost-effective, advantageous and superior to liquid priming as it is convenient to any seed size, very less amount of liquid is required (thus this is economical) and the slow absorption of liquid in this method allows to repair the damages, if any, occurred during liquid uptake (very fast liquid uptake in liquid priming frequently causes injury to cotyledons). Hence, farmers should be encouraged to use it on an extensive basis. Moreover, going with the modern approach of seed nano-priming, assessment of the potential role of nano-chitosan as priming agent on mung bean is not investigated yet. Synthesizing novel nanoparticles like nano-chitosan using organic, biodegradable basic materials is gaining popularity because of its prospects for economical, ecologically friendly, and mass production. In the current study, after the synthesis of nano-chitosan using biodegradable chitosan as base material through the ionic gelation method in the laboratory, it was physiochemically characterized through Dynamic Light Scattering (DLS) and zeta potential studies, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray analysis (EDX), and Fourier Transmission Infrared Spectroscopy (FTIR). The prepared nano-chitosan was then applied through SMP in mung bean under different concentrations of salinity stress to investigate its effect on the various seedling growth parameters, their biochemical and enzymological attributes, and salt-induced oxidative stress mitigation. The antioxidant activities of the treated mung bean seedlings under the said conditions were also studied. SMP noticeably enhanced the activities of antioxidant enzymes, namely Catalase (CAT), Peroxidase (POD), Superoxide dismutase (SOD), Polyphenol Oxidase (PPO), Ascorbate peroxidase (APX) activities in treated mung bean seedlings. At the same time, significant enhancement in the nutraceutical values of mung bean sprouts were also noted. Further, it noticeably reduces oxidative damages along with noteworthy improvement in terms of tolerance capacity in mung bean seedlings exposed to salinity stress conditions. The efficacy of nano-chitosan was also assessed for its antifungal activity against Aspergillus flavus, the most common mung bean seed-borne mycoflora, identified by the Indian Agricultural Research Institute (IARI), Delhi, India. Fungal spore germination and mycelial growth were rigorously examined after applying nano-chitosan in a dose-dependent manner with sterilization procedure modification. To evaluate the effect of nano-chitosan following mung bean seed priming (SMP), Aspergillus flavus spore suspension was added to the seeds and allowed to germinate. After seven days of germination, various growth parameters of mung bean seedlings were measured, showing the highest antifungal activities with 571 mg/ml of nano-chitosan at a 10% matrix (Celite) moisture level. Accordingly, the current study revealed that SMP with nano-chitosan is an effective seedling invigoration treatment in saline settings, especially for mung bean seedlings in their early growth stages. Thereafter, the treated and untreated mung bean seedlings under salt stress conditions were subjected to On-gel isozyme patterns of Native PAGE (α-Amylase, NADPH Oxidase, Superoxide dismutase, Peroxidase) and SDS-PAGE. Orbitrap High-Resolution Liquid chromatography-mass spectrometry (OHR-LCMS) was conducted from SAIF, IIT- Bombay, to examine mung bean seedlings' most prominent and variably expressed SDS gel band particularly obtained in lane 3 represented by nano-chitosan treated seedlings in the gel image. The High-Resolution Liquid chromatography-mass spectrometry (HR-LCMS) analysis of three significant isoflavones (Genistein, Formononetin, and Biochanin A) in control and chitosan and nano-chitosan treated mung bean seedlings grown under salt-stress conditions was conducted separately from SAIF, IIT-Bombay. Further, the major mung bean isoflavones identified by the HR-LCMS study, were undergone through in-silico drug study (ADME and druggability test, prediction of pharmacological activities through PASS), and toxicity analysis (PRED SKIN, Pred-hERG Analysis, CarcinoPred-EL, Xenosite reactivity) using various online web tools in connection with their associated therapeutic implications. STRING and Cytoscape analysis were also performed to realize the interaction between the isoflavones and the salt stress genes in mung bean. The metabolic shifts of chief isoflavones having therapeutic consequences were critically detected. Further, to evaluate the impact of SMP with nano-chitosan in the genetic expression of some selected salt stress genes (SOS1, SOS2, SOS3, and NHX1) of mung bean, Quantitative Real Time PCR analysis of the treated (nano-chitosan primed) and untreated (control) mung bean seedlings was conducted from Credora, Life Sciences, Bengaluru, Karnataka. In every case, distinctly significant and enhanced expression of the selected salt stress genes were remarkably noted in the nano-chitosan treated seedlings compared to the untreated ones grown under salinity stress conditions. This clearly suggests that nano-chitosan (applied through SMP), is an ideal primer for seedling invigoration and salinity induced oxidative stress mitigation. So, mung bean sprouts can be considered as a well-known healthier alternative and because of their prospective impacts on human healthiness and the industry's rapid growth, nutritional fortification of this functional food has become an important topic of study. Moreover, this work on mung bean may shed light on the functions of some associated salt-tolerant genes and the underlying molecular mechanisms of salt tolerance in mung bean crop plants. Thus, in modern agricultural system, application of nano-chitosan through SMP might be a promising technique in stress mitigation, along with protecting and invigorating crop plants like mung bean against abiotic stress like salinity which in turn gives a conceptual foundation for the future study on the strengthening of mung bean's salt tolerance mechanism and the genetic resources. Further, based on the valuable characteristics like, antifungal and antioxidant activity, biodegradable and biocompatible nano-chitosan might be a potential substitute in place of commonly used toxic and non-degradable agrichemicals especially for the targeted beneficiaries and would also be helpful for the society and the environment in the long run.
Open Access
Studies on the occurrence of antibiotic resistance and virulence in motile Aeromonas species from fish farming environments in sub-Himalayan West Bengal
(University of North Bengal, 2022) Mangar Preeti; Saha Aniruddha
Fisheries is an important source of commercial export in India and serves as a means of livelihood for the people at large. In India, West Bengal is a one of the leading producers of fish owing to its optimum agroclimatic conditions and inhabitants’ ingrained fondness for fish. Being placed at the foot hills of the great Himalayas, North Bengal holds unique endemism and icthyodiversity which set it as a class apart from rest of the state. Fishes are sometimes reared in small scale in homestead ponds, which forms the alternative sources of income among the rural population. However, fishes are reared without proper farm practices which leads to disease outbreaks with ultimate impact on the productivity. The genus Aeromonas comprises of gram-negative bacteria widely disseminated in the aquatic environments. Mostly the members of these genus are opportunistic pathogens and capable of inducing diseases within a wide host range. Various researchers have reported the presence of these microorganisms in Epizootic-Ulcerative syndrome and haemorrhagic lesions in the fishes in Northern part of West Bengal. The primary goal of the present study comprised of analyzing the prevalence of Aeromonas sp. from water samples collected from fish farming environments of three districts of North Bengal. Secondly, the aeromonads were screened for virulence factors and its encoding genes responsible for pathogenic invasion of host. Thirdly, due to its abundance, the isolated aeromonads were evaluated for resistance against commonly used antibiotics and investigated for the underlying resistance genes. The objectives of the present research were: (i) Isolation of Aeromonas spp. from fish farming environments in sub-Himalayan West Bengal. (ii) Biochemical characterization and phylogenetic analysis of bacterial isolates based on 16S rRNA gene sequence. (iii) Study of the virulence properties of the isolates of Aeromonas strains. (iii) Antibiotic sensitivity profiling of the bacterial isolates. (iv) Analyzing bacterial DNA for the presence of genetic determinants of resistance and virulence. (v) Study the mobility of antibiotic resistance coding genes by in vitro conjugation. For fulfilment of the objectives, water samples were collected from ten different small fish farming ponds distributed across three districts (Darjeeling, Jalpaiguri and Coochbehar) of North Bengal. The samples were subjected to microbiological processing which led to the isolation of total 83 putative Aeromonas strains in Aeromonas isolation medium. All these isolates were further screened by following the modified scheme of biochemical identification known as Aerokey-II which led to the identification of 34 strains as Aeromonas sp. The isolates were further confirmed to belong to this genus by PCR methods. The16S rRNA gene sequencing and BLAST similarity search revealed that the strains matched Aeromonas with percentage identity of the isolates as > 98%. The gene sequences were subjected to phylogenetic characterization and all the isolates rearranged to four reference strains: A. veronii (n=19), A. hydrophila (n=7), A. jandei (n=5) and A. caviae (n=3). The sequences were deposited in NCBI GenBank, and accession numbers were assigned to all the thirty-four isolates, viz. A. veronii (MT378391, MT379551, MT379645, MT379646, MT385145, MT380477, MT383121, MT383124, MT384421, MT384337, MT385097, MT396085, MT393933, MT393944, MT396230, MT396438, MT397061, MT397058, MT397063), A. hydrophila (MT378381, MT379550, MT379552, MT396436, MT396445, MT396437, MT395673), A. jandei (MT378390, MT393937, MT393941, MT393945, MT393942), A. caviae (MT393443, MT393930, MT393932). The pathogenicity in Aeromonas is multifactorial and linked to a large number of genes that encode for various toxins and structural elements which aids proliferation within the host. In this study, six important virulence factors, hemolysin, protease, lipase, amylase, DNase and siderophore production were tested in all the 34 identified isolates of Aeromonas. Among the tested virulence traits, hemolytic and proteolytic activity was exhibited by 100% of the isolates. Amylase was detected in 70.6% of the isolates and DNase activity was detected in 44.10% of the isolates. Siderophore was detected in 38.20% of the total isolates and a very small proportion of only 5.80% of isolates showed lipase activity. Further, four important virulence genes aer/haem (encoding hemolysin), aspA (encoding alkaine serine protease), ascV (encoding type 3 secretion system) and flaA (encoding polar flagella) were analysed in all the 34 isolates identified as Aeromonas. PCR amplification of the genes revealed that 44.11% of the Aeromonas isolates carried the aer/haem and flaA genes. The ascV gene was found in 23.5% of the isolates. Only 8.82% of the aeromonads contained the aspA gene. The amplification products were cloned and sequenced. The sequences were subjected to similarity search using the BLASTn tool, annotated and submitted to the NCBI database through the BANKIT tool. The virulence genes have the following GenBank accession numbers: aer/haem (MT704303-MT704309; MT707932-MT707935; MH607886, MT591426, and MTT813045) aspA (MT909568-MT909570); ascV (MW001219-MW001222; MH607887-MH607890); and flaA (MT942623-MT942626, MT977537- MT977539). Based on the combination of virulence genes found to be present in the Aeromonas isolates, the strains were classified into nine genotypic groups. Most of the Aeromonas strains belonged to group G that harboured only the flaA+ gene. The second common genotype among the isolates was aer/haem+, ascV+, flaA+. This study is the first to report the presence of such virulence genes from aeromonads isolated from this region. Furthermore, a virulent isolate of Aeromonas GP3 was able to transfer the aer/haem gene to E. coli DH5α via conjugation with an efficiency of 0.0394 X10-4 transconjugants per recipient cell. The detection of 23 kb plasmids in both donor and transconjugants corroborated to the transfer and gave an insight of the aer/haem being plasmid borne. In order to validate the pathogenicity of the aeromonads six virulent isolates GP3, RB7, BP3, RJB1, MG8 and PP21 from different sampling sites and harboring atleast two virulence traits were injected into Anabas testudineus. The most harmful strain was GP3, which also possessed three of the four tested virulence-related genes (aer/haem+/ ascV +/flaA+) and five virulence features (hemolysin, protease, DNase, siderophore, and amylase). On the other hand, strain PP21 which showed only two pathogenic phenotypes (protease and hemolysin) and the genes aerA/haem+ and flaA+, also showed 100% mortality. Contrarily, RB7, which exhibited four pathogenic phenotypes (hemolysin, protease, DNase, and amylase) and three genes (aerA/haem+/ascV+/aspA+), did not induce any mortality. In addition, BP3 was found to be very harmful to fishes despite only carrying one of the four genes under investigation which was ascV and exhibited four virulence characteristics. Therefore, in the current study it may be concluded that virulence phenotype expression and fish mortality were unrelated. The isolated virulent Aeromonas sp. was further studied for its cytotoxicity in WRL-68 cell line (human, liver, embryonic). Results of this study revealed that the cell free culture filtrates of the Aeromonas strain GP3 were cytotoxic to human liver cells. Microscopic observation of the cells showed visible rounding off and detachment from the surface when compared against control cells, treated with cell free culture filtrate of Lactobacillus sp. (non-pathogen) with minor changes in morphology. The cell viability was reduced to 0.48% in GP3 filtrate treated cells as compared to 66% viability in Lactobacillus sp. treated cells. Hence the cytotoxic effect of GP3 on human cell lines implicating its pathogenic potential has been well established in this study. In the current study, the partial nucleotide sequence of hemolysin amplified by PCR from GP3 was found to encode 185 amino acids. The 3D structure of the translated protein was constructed by homology modelling using a reference template 3COM (pro aerolysin of Aeromonas hydrophila) showing >90% homology. Further validation was done by PROCHECK software. Such modelling of the protein structure could prove to be beneficial for vaccine development and drug targeting of pathogenic aeromonads. With the rise of diseases which hampers the fish productivity, numerous antibiotics are being used both as therapeutics and prophylactics that often leads to resistance among the bacterial populations. In this study resistance of the 34 isolates against 20 commonly used antibiotics for treatment of diseases were assessed. In the current study 100% resistance was observed against penicillin and ampicillin followed by imipenem (85.29%), streptomycin (58.82%), cefoperazone (52.94%) and trimethoprim (50%). All the tested aeromonads exhibited resistance to three or more antibiotics. Intermediate level of resistance was observed against cefepime and cefuroxime (38.24%) and cefepime (23.53%). Maximum level of sensitivity was observed against chloramphenicol (91.18%) followed by norfloxacin (82.35%). Very high multiple antibiotic resistance (MAR) indices ranging between 0.15–0.7 in 94.1% of the isolates were observed which indicated that the aeromonads in fish farming environments have been exposed to antimicrobials. A MAR index > 0.2 indicates an environment with continuous exposure to antibiotics. A particular Aeromonas strain MG8 showing resistance against 14 antibiotics was found. Two strains, MG3 and HP6 showed resistance to 11 antibiotics, while strain PP19 showed resistance to 10 antibiotics. Six significant clusters (C1-C6) were found using the Wards minimum distance approach in the hierarchical cluster analysis based on the antibiotic susceptibility phenotype represented as zone widths and sample sites of the 34 Aeromonas strains. The strains from different locations grouped to some extent under similar cluster depending on their levels of antibiotic resistance, like, all the Shivmandir isolates with low average MAR index value grouped in C2. However, variations in antibiotic resistance phenotype within a particular location was found to be the predominant feature. Therefore, it was concluded that resistance in aeromonads is irrespective of the location of sampling and the isolates had strain specific resistance pattern. In the current study, genetic elements encoding antibiotic resistance known as integrons were screened for their presence in all 34 isolates. class I integrons (intI) were detected in 38.23% of the isolates with sizes ranging from 0.65 kb-2.5 kb. The integrons of 1.4 kb detected in FP2, MG8 and HP6 were cloned and sequenced. The NCBI accession numbers are MT424748, OP610544 and OP745416 respectively. The 1400 bp integron cassette of FP2 revealed the insertion of resistance gene cassettes of aadA4 and qacE providing resistance against aminoglycosides and quaternary ammonium compounds respectively. The integron cassette of MG8 with a size of 1400 bp had the resistance gene casettes dhfrA1 and aadA1 providing resistance against sulfonamides and aminoglycosides respectively. The integron size of 1400 bp in isolate HP6 possessed dhfrA showing resistance against sulfonamides and a hypothetical protein of unknown function. Similarly, tetracycline resistance gene tetE encoding an efflux pump was detected in one isolate PP23 and the GenBank accession no. is given as OP745417. Plasmids of sizes ranging between 1.6kb – 23 kb were present in a few isolates (PP7, RB5, GP1, MG9 and BP5) and all these plasmids were transferable to E. coli Dh5α by in-vitro conjugation experiments. Four isolates were able to transfer their 23 kb plasmid to E. coli Dh5α and the resistance markers of cefepime, cefuroxime, ampicillin, and oxytetracycline resistance could be traced in the recipients as well. Similarly, an isolate BP5 was capable of transferring 4.3kb and 1.6kb plasmids to the E. coli DH5α via conjugation. Conjugational transfer frequencies of 0.15 X 10-7 - 0.76 X 10-6 were obtained. In conclusion, the current study reports the prevalence of Aeromonas sp. in small fish farming environments. The major findings of this study were the detection of virulence and antibiotic resistance in aeromonads along with underlying genes from three districts of West Bengal. The study has given a current scenario of the level of contamination of the fish farms by opportunistic pathogens like Aeromonas sp. The study may be helpful in spreading awareness among the fish farmers to adopt proper practices and avoid the disease outbreaks. Also, a clear picture regarding the level of antibiotic resistance in fish farms gives a picture about misuse of antibiotics. The data could be utilized to educate small scale farmers regarding the controlled use of drugs and antibiotics in fish culture.

Department of Botany

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