Papers by Bidhan Dhar
Microorganisms, Feb 12, 2024
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Microorganisms, Feb 12, 2024
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Frontiers in Horticulture, Mar 16, 2023
With the goal to develop management tools to mitigate the severity of shoot blight on pear caused... more With the goal to develop management tools to mitigate the severity of shoot blight on pear caused by Erwinia amylovora and prevent fire blight canker development from infected shoots on pear tree wood, we evaluated five preventive treatments in inoculated experiments over 2-years. We focused on foliar spray and trunk injection applications of extract of giant knotweed, Reynoutria sachalinensis (RSE), as a plant resistance activator candidate and on comparisons to antibiotics. Five foliar applications of RSE (2.24 L/ha) on 6-yearold trees of cultivar 'Bartlett' resulted in 100% control of shoot blight severity and canker incidence in both years of evaluation. The RSE trunk injection in fall of the first year did not control the disease allowing 75% of shoot severity and 38.5% of canker incidence. The opposite happened in the second year, when we injected RSE in spring, providing 85.7% control of shoot blight severity and 89.8% control of canker incidence. Antibiotics oxytetracycline (OTC) and streptomycin (STM) did not provide consistent control in the second year in comparison to the first year. In the first year, both antibiotics, OTC (trunk injection and foliar spray) and STM were effective, providing 94.8%, 100% and 86.3% control of shoot blight, respectively. They also provided 100, 91 and 84% control of canker incidence, respectively. However, in the second they were not effective allowing 69-96% of shoot blight severity and 70-92% of canker incidence. Regardless of the inconsistency in the effectiveness of antibiotics, which are not recommended for shoot blight control due to risks of promoting antibiotic resistance, five preventive spray applications of RSE could be used on Frontiers in Horticulture frontiersin.org 01
Analytical and Bioanalytical Chemistry, 2022
International Journal of Environmental Research and Public Health, 2015
International Journal of Environmental Research and Public Health, 2015
Monitoring the quality of water is of paramount importance for public health. According to the Wa... more Monitoring the quality of water is of paramount importance for public health. According to the Water Framework Directive 2000/60/EC, “water is not a commercial product but a heritage that must be protected, defended and treated as such”. In Europe, about 40% of the drinking water is derived from surface waters and pathogenic organisms occurring in lakes and rivers represent a particularly serious health-hazard. In fact, approximately 100 million Europeans lack safe and reliable water supplies and microbial pathogens in drinking water are causing significant morbidity and mortality with >170.000 estimated cases of water-related disease in Europe (UN News Centre, Protocol on Water and Health). The potential threat of water contamination and the spread of waterborne diseases are becoming more serious than ever before. This is the result of a number of factors, including the increase in population, globalisation and the movement of more people across borders and between continents, a...
Critical Reviews in Environmental Science and Technology, 2021
Biosensors and Bioelectronics
Abstract: A few diatom species produce toxins that affect human and animal health. Among these, m... more Abstract: A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan
shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based
on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker
genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic
acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets.
The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of
this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.
Our understanding of the composition of diatom communities and their response to environmental ch... more Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms.
There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic
polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting
eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.
Thesis Chapters by Bidhan Dhar
Monitoring the quality of water is of paramount importance for public health. According to the Wa... more Monitoring the quality of water is of paramount importance for public health. According to the Water Framework Directive 2000/60/EC, “water is not a commercial product but a heritage that must be protected, defended and treated as such”. In Europe, about 40% of the drinking water is derived from surface waters and pathogenic organisms occurring in lakes and rivers represent a particularly serious health-hazard. In fact, approximately 100 million
Europeans lack safe and reliable water supplies and microbial pathogens in drinking water are causing significant morbidity and mortality with >170.000 estimated cases of water-related disease in Europe (UN News Centre, Protocol on Water and Health). The potential threat of
water contamination and the spread of waterborne diseases are becoming more serious than ever before. This is the result of a number of factors, including the increase in population, globalisation and the movement of more people across borders and between continents, and the effects of global warming. Furthermore, both abundance and distribution of all microbes are expected to change as a result of global climate change that will cause, for instance, increased or decreased rainfall, temperature and radiation.
As the nature of the diatom communities reflects environmental conditions and since they react rapidly and sensitively to water quality changes, these microorganisms have become widely used over the last 50 years as biomarkers for water quality assessment. Moreover, diatom doubling time is one of the quickest among various biological indicators and rapidly reveals a change in water quality. Diatoms are excellent bioindicators [...]. Traditional methods for diatom classification are based on microscopic frustule morphology but are laborious, time-consuming and not suitable for routine monitoring programs. Molecular biological tools have now greatly enhanced our ability to investigate biodiversity by identifying species and estimating gene flow and distribution of species.
Therefore, there is a strong incentive to explore and develop alternative methods that are faster, less expensive, reliable and efficient for identification of diatoms in complex environmental samples.
Microarrays have been the subject of several EU projects, like EU PICODIV and MICROPAD. Microarrays were developed for the detection of algae and protozoa, and results
from chip hybridization were favourably compared to other measurements of diversity, (i.e., direct cell counts and clone libraries). In these two projects, the microarrays were in early stages of development and proof of principle was the major outcome, because it was discovered that probes previously made for fluorescence in situ hybridisation (FISH) could not be directly transferred to a microarray chip format. With a few exceptions, nearly every probe had to be modified for successful use in the microarray chip format. Problems with transferring FISH probes to a microarray chip format led workers in the EU project MIDICHIP to modify their probes and microarrays, especially those designed for detection of cyanobacteria. The FP6 project FISH AND CHIPS made use of prototype findings to develop a microarray chip for phytoplankton at the class level and field data were analysed over three years with rRNA as the preferred target molecule [...] In the EU project AQUACHIP, pathogenic bacteria were the target of interest. This project developed a chip for five bacteria but they were not widely tested with environmental samples. In addition, the detection system developed for this chip was based on a microtiter plate system with detection under a fluorescent microscope. This, however, is not a standard protocol that can be used in a commercial microarray chip reader.
The results obtained in the earlier European Union (EU) projects were promising and contributed to the success of an application for an EU-funded grant. μAQUA is the acronym
for the EU research project "Universal microarrays for the evaluation of fresh-water quality based on detection of pathogens and their toxins" funded by the 7th Framework Programme of the European Commission and coordinated by Dr. Claudio Gualerzi and Dr. Roberto Spurio of the University of Camerino (Italy). μAQUA aims to design and develop a universal microarray chip for the high-throughput detection in freshwater of known and emerging pathogens (bacteria, viruses, protozoa and cyanobacteria) that cause human diseases and to assess the water quality by monitoring the presence of select bioindicators (diatoms). Existing
techniques are laborious and time-consuming, requiring labor-intensive cultivation and microscopic examination of potential pathogens from water samples. The innovative
molecular biological techniques being investigated by the μAQUA, by contrast, should enable the rapid - and more reliable - detection of pathogens and diatoms in large volumes of water.
The design of probes specifically targeting rDNA or rRNA from higher rank down to species level is made possible by the steadily growing number of rDNA-sequences available
(e.g. in the Ribosomal Database Project) [...]. The rDNAspecific probes are used to analyse phytoplankton communities with detection by flow cytometry, epifluorescence microscopy [...] or other methods that take advantage of the specificity ensured by nucleic acid hybridisation. However, a major drawback of these methods is that they are very time-consuming vis-à-vis the complexity of a microbial sample insofar as they can only be used to identify one or few organisms at a time [...]. By contrast, the DNA-microarray chip technology, which allows the rapid and simultaneous analysis of up to 200,000 probes without a cultivation step [...], has enormous potential as a method of choice for the analysis of complex environmental samples
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Papers by Bidhan Dhar
shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based
on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker
genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic
acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets.
The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of
this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.
There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic
polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting
eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.
Thesis Chapters by Bidhan Dhar
Europeans lack safe and reliable water supplies and microbial pathogens in drinking water are causing significant morbidity and mortality with >170.000 estimated cases of water-related disease in Europe (UN News Centre, Protocol on Water and Health). The potential threat of
water contamination and the spread of waterborne diseases are becoming more serious than ever before. This is the result of a number of factors, including the increase in population, globalisation and the movement of more people across borders and between continents, and the effects of global warming. Furthermore, both abundance and distribution of all microbes are expected to change as a result of global climate change that will cause, for instance, increased or decreased rainfall, temperature and radiation.
As the nature of the diatom communities reflects environmental conditions and since they react rapidly and sensitively to water quality changes, these microorganisms have become widely used over the last 50 years as biomarkers for water quality assessment. Moreover, diatom doubling time is one of the quickest among various biological indicators and rapidly reveals a change in water quality. Diatoms are excellent bioindicators [...]. Traditional methods for diatom classification are based on microscopic frustule morphology but are laborious, time-consuming and not suitable for routine monitoring programs. Molecular biological tools have now greatly enhanced our ability to investigate biodiversity by identifying species and estimating gene flow and distribution of species.
Therefore, there is a strong incentive to explore and develop alternative methods that are faster, less expensive, reliable and efficient for identification of diatoms in complex environmental samples.
Microarrays have been the subject of several EU projects, like EU PICODIV and MICROPAD. Microarrays were developed for the detection of algae and protozoa, and results
from chip hybridization were favourably compared to other measurements of diversity, (i.e., direct cell counts and clone libraries). In these two projects, the microarrays were in early stages of development and proof of principle was the major outcome, because it was discovered that probes previously made for fluorescence in situ hybridisation (FISH) could not be directly transferred to a microarray chip format. With a few exceptions, nearly every probe had to be modified for successful use in the microarray chip format. Problems with transferring FISH probes to a microarray chip format led workers in the EU project MIDICHIP to modify their probes and microarrays, especially those designed for detection of cyanobacteria. The FP6 project FISH AND CHIPS made use of prototype findings to develop a microarray chip for phytoplankton at the class level and field data were analysed over three years with rRNA as the preferred target molecule [...] In the EU project AQUACHIP, pathogenic bacteria were the target of interest. This project developed a chip for five bacteria but they were not widely tested with environmental samples. In addition, the detection system developed for this chip was based on a microtiter plate system with detection under a fluorescent microscope. This, however, is not a standard protocol that can be used in a commercial microarray chip reader.
The results obtained in the earlier European Union (EU) projects were promising and contributed to the success of an application for an EU-funded grant. μAQUA is the acronym
for the EU research project "Universal microarrays for the evaluation of fresh-water quality based on detection of pathogens and their toxins" funded by the 7th Framework Programme of the European Commission and coordinated by Dr. Claudio Gualerzi and Dr. Roberto Spurio of the University of Camerino (Italy). μAQUA aims to design and develop a universal microarray chip for the high-throughput detection in freshwater of known and emerging pathogens (bacteria, viruses, protozoa and cyanobacteria) that cause human diseases and to assess the water quality by monitoring the presence of select bioindicators (diatoms). Existing
techniques are laborious and time-consuming, requiring labor-intensive cultivation and microscopic examination of potential pathogens from water samples. The innovative
molecular biological techniques being investigated by the μAQUA, by contrast, should enable the rapid - and more reliable - detection of pathogens and diatoms in large volumes of water.
The design of probes specifically targeting rDNA or rRNA from higher rank down to species level is made possible by the steadily growing number of rDNA-sequences available
(e.g. in the Ribosomal Database Project) [...]. The rDNAspecific probes are used to analyse phytoplankton communities with detection by flow cytometry, epifluorescence microscopy [...] or other methods that take advantage of the specificity ensured by nucleic acid hybridisation. However, a major drawback of these methods is that they are very time-consuming vis-à-vis the complexity of a microbial sample insofar as they can only be used to identify one or few organisms at a time [...]. By contrast, the DNA-microarray chip technology, which allows the rapid and simultaneous analysis of up to 200,000 probes without a cultivation step [...], has enormous potential as a method of choice for the analysis of complex environmental samples
shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based
on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker
genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic
acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets.
The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of
this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.
There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic
polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting
eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.
Europeans lack safe and reliable water supplies and microbial pathogens in drinking water are causing significant morbidity and mortality with >170.000 estimated cases of water-related disease in Europe (UN News Centre, Protocol on Water and Health). The potential threat of
water contamination and the spread of waterborne diseases are becoming more serious than ever before. This is the result of a number of factors, including the increase in population, globalisation and the movement of more people across borders and between continents, and the effects of global warming. Furthermore, both abundance and distribution of all microbes are expected to change as a result of global climate change that will cause, for instance, increased or decreased rainfall, temperature and radiation.
As the nature of the diatom communities reflects environmental conditions and since they react rapidly and sensitively to water quality changes, these microorganisms have become widely used over the last 50 years as biomarkers for water quality assessment. Moreover, diatom doubling time is one of the quickest among various biological indicators and rapidly reveals a change in water quality. Diatoms are excellent bioindicators [...]. Traditional methods for diatom classification are based on microscopic frustule morphology but are laborious, time-consuming and not suitable for routine monitoring programs. Molecular biological tools have now greatly enhanced our ability to investigate biodiversity by identifying species and estimating gene flow and distribution of species.
Therefore, there is a strong incentive to explore and develop alternative methods that are faster, less expensive, reliable and efficient for identification of diatoms in complex environmental samples.
Microarrays have been the subject of several EU projects, like EU PICODIV and MICROPAD. Microarrays were developed for the detection of algae and protozoa, and results
from chip hybridization were favourably compared to other measurements of diversity, (i.e., direct cell counts and clone libraries). In these two projects, the microarrays were in early stages of development and proof of principle was the major outcome, because it was discovered that probes previously made for fluorescence in situ hybridisation (FISH) could not be directly transferred to a microarray chip format. With a few exceptions, nearly every probe had to be modified for successful use in the microarray chip format. Problems with transferring FISH probes to a microarray chip format led workers in the EU project MIDICHIP to modify their probes and microarrays, especially those designed for detection of cyanobacteria. The FP6 project FISH AND CHIPS made use of prototype findings to develop a microarray chip for phytoplankton at the class level and field data were analysed over three years with rRNA as the preferred target molecule [...] In the EU project AQUACHIP, pathogenic bacteria were the target of interest. This project developed a chip for five bacteria but they were not widely tested with environmental samples. In addition, the detection system developed for this chip was based on a microtiter plate system with detection under a fluorescent microscope. This, however, is not a standard protocol that can be used in a commercial microarray chip reader.
The results obtained in the earlier European Union (EU) projects were promising and contributed to the success of an application for an EU-funded grant. μAQUA is the acronym
for the EU research project "Universal microarrays for the evaluation of fresh-water quality based on detection of pathogens and their toxins" funded by the 7th Framework Programme of the European Commission and coordinated by Dr. Claudio Gualerzi and Dr. Roberto Spurio of the University of Camerino (Italy). μAQUA aims to design and develop a universal microarray chip for the high-throughput detection in freshwater of known and emerging pathogens (bacteria, viruses, protozoa and cyanobacteria) that cause human diseases and to assess the water quality by monitoring the presence of select bioindicators (diatoms). Existing
techniques are laborious and time-consuming, requiring labor-intensive cultivation and microscopic examination of potential pathogens from water samples. The innovative
molecular biological techniques being investigated by the μAQUA, by contrast, should enable the rapid - and more reliable - detection of pathogens and diatoms in large volumes of water.
The design of probes specifically targeting rDNA or rRNA from higher rank down to species level is made possible by the steadily growing number of rDNA-sequences available
(e.g. in the Ribosomal Database Project) [...]. The rDNAspecific probes are used to analyse phytoplankton communities with detection by flow cytometry, epifluorescence microscopy [...] or other methods that take advantage of the specificity ensured by nucleic acid hybridisation. However, a major drawback of these methods is that they are very time-consuming vis-à-vis the complexity of a microbial sample insofar as they can only be used to identify one or few organisms at a time [...]. By contrast, the DNA-microarray chip technology, which allows the rapid and simultaneous analysis of up to 200,000 probes without a cultivation step [...], has enormous potential as a method of choice for the analysis of complex environmental samples