Department of Botany

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    ItemOpen 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?
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    ItemOpen 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.
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    ItemOpen Access
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    ItemOpen 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.
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    ItemOpen 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.
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