Stress tolerance in bacterial strains of the genus Rhodanobacter isolated from a mixed waste contaminated subsurface

Oak Ridge Integrated Field Research Challenge (ORIFRC) site is characterized by low pH and consists of high nitrate, organics and heavy metals. ORIFRC field laboratory comprises variety of contaminants (uranium, technetium, nitrate, volatile organic carbon species etc.) which are of interest to US Department of Energy. Rhodanobacter is a dominant bacterial species found at this site and ideal for remediation of such mixed contaminated sites.

A collaborative study was conducted by researchers at Florida State University, NCMR-NCCS Pune, University of Illinois, Georgia Institute of Technology and Symbiosis School of Biological Sciences to understand the physiologic basis of stress tolerance in members of the genus Rhodanobacter. The study was conducted in order to understand how bacterial strains of the genus Rhodanobacter survive and dominate in the mixed waste contaminated habitats of the ORIFRC site. To address this, a systematic analysis of relevant phenotypic properties of strains of the genus Rhodanobacter was studied.

Eight strains of Rhodanobacter were isolated from high and low contaminated zones and used for pH and nitrate utilization studies. NaCl, nitrate, nitrite and heavy metal tolerance capacity was studied for the two selected strains of R. denitrificans. Based on metals known to be present at the ORIFRC site, Rhodanobacter strains were tested for tolerance to zinc, cadmium, cobalt, nickel, copper and uranium. To determine the effect of incubation time on growth of R. denitrificans at high metal concentrations, studies were carried out with nickel and uranium since these two metals were found to be present at very high concentrations at the test site.

The results supported the growth potential of Rhodanobacter in acidic subsurface groundwater conditions and confirmed that under suitable cultivation conditions, isolated R. denitrificans strains can tolerate acidic pH consistent with ORIFRC site pH values. It was also observed that organisms adapted to stress better under conditions of high organic content. The ability of Rhodanobacter strains to grow at extremely low pH and under high nitrate and heavy metals concentrations is responsible for their dominance at the contaminated subsurface of the ORIFRC site. The data indicated that both the strains are well adapted to the eco physiological conditions of the contaminated ORIFRC site.

As bacteria from the genus Rhodanobacter are denitrifiers, their activity in the ORIFRC site subsurface is also linked to carbon and nitrogen cycling and may play a critical role in the bioremediation of uranium. Based on prior findings and the results of the current study, researchers postulated that low pH tolerance and high level of stress tolerance for a range of metals along with denitrification potential gives a selective advantage to members of the genus Rhodanobacter. Bacteria from the genus Rhodanobacter are facultative anaerobes, and this physiologic capability makes them ideal candidates for robust growth in the contaminated subsurface of the ORIFRC site. Due to their enhanced stress tolerance abilities, Rhodanobacter spp. survives at low pH and in the presence of elevated concentrations of heavy metals, nitrate and nitrite.


PAN-India SARS-CoV-2 genome sequencing reveals important insights into the outbreak

The ongoing pandemic of Severe Acute Respiratory Syndrome (SARS-CoV-2) has emerged as a global health problem and has adversely affected the world. The novel corona virus is spreading rapidly creating a threat for humankind. It is estimated that it can spread twice as fast as the 1918 Spanish flu virus. Whole-genome sequencing of pathogens, especially viruses, is a powerful tool to generate rapid information on outbreaks. The results from this technique help in effective understanding of the introduction of the infection ,dynamics of transmission, contact tracing networks and impact of informed outbreak control decisions. This technique has been effective in earlier outbreaks like the Ebola virus.

A collaborative study was conducted by researchers from NIBMG Kalyani, ILS Bhubaneswar, CDFD Hyderabad, NCBS Bengaluru, InStem Bengaluru, NCCS Pune and ICMR-Regional Centre for Medical Research, Bhubaneswar in order to achieve initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes. The nasal and oral swabs were collected from individuals testing positive for COVID-19. The samples were collected from 10 states covering different zones within India. Phylodynamic analysis, mutation analysis and haplotype network analysis was performed. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. This study will contribute in understanding how the virus is spreading, ultimately helping to restrict transmission, prevent new cases of infection, and provide information for research on how to interevent the spread of infection.

Preliminary results indicated that multiple lineages of SARS-CoV-2 are circulating in India, might have introduced by travel from Europe, USA and East Asia. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Scientists were able to estimate the possible source of country of different varieties of the virus introduced in India because of travel. The virus has also mutated and one of the mutations has attained highest frequencies across most of the states. There are two lineages of the virus named as 20A and 20B which are predominant across the country. The haplotype 20A is most abundant in northern and eastern India, 20B haplotype was abundant in southern and western India. The ancestral haplotypes of 19A and 19B were mostly found in Northern and Eastern India, with 19B being the most abundant in the latter region.

Analysis indicated that the haplotype diversities across India and in each region continued to increase until May 2020, after that it reduced drastically with the emergence of the A2a haplotypes which has overtaken other lineages by June 2020. Such interpretations might enable improved understanding of the virus and hence the health decisions. From the haplotype network, researchers observed that Maharashtra, Karnataka created three distinct haplotype nodes and sequences from Odisha, West Bengal and Uttarakhand sparse in different haplotype nodes. They also observed a haplotype node with the majority of the genomes from West Bengal, Odisha and a small percentage of the samples belonging to Uttarakhand.

Analysis of probable country of origin of these SARS-CoV-2 sequences in India revealed that they had been probably introduced by travel from multiple countries across the globe. 20A, B and C haplotypes were introduced from multiple countries in Europe and also American continents. Interestingly, 20A alone is predicted to have been introduced by travel from Italy, Saudi Arabia, United Kingdom and Switzerland. Similarly, 20B was introduced from the United Kingdom, Brazil, Italy and Greece. In contrast, 19A was introduced from China alone while 19B was introduced by travel from China, Oman and Saudi Arabia.

The number of COVID-19 occurrences in India has increased drastically over the time. Although most of the states have their own strategic lockdown devised to control the outbreak, it will be more efficient if we can include the geographical transmission pattern information in the planning of such strategies. In the current study, scientists have tried to explore the transmission of the infection among different states of India. It is necessary to add more genomic datasets to understand clear picture.


Meet Dr. Neetha Joseph- Scientist at NCMR-NCCS Pune

Dr. Neetha Joseph’s research interest is in microbial systematics, ecology and community analysis. She is affiliated with NCMR-NCCS Pune from last 8 years. She is in-charge of FAME analysis service and curator of Firmicutes. It was a great pleasure to interact with Dr. Neetha and to know more about her as a person and her work.

Kranti: Dr. Neetha, you have worked with coastal environment micro-organisms during your PhD. At a personal level, what motivated you to enter into microbiology research?
Dr. Neetha: Kranti, my native place is in Kerala, a beautiful coastal area in India. Kochi is a lovely place with lot of Backwaters and Estuaries. When I finished my post-graduation, I got an opportunity to join at National Institute of Oceanography (NIO) where most of the research work is related to Ocean and Estuaries. Nutrient enrichment due to various anthropogenic activities is the most widespread problem in estuaries around the world. Significant spatial and temporal variability of physico-chemical and geochemical characteristics and productivity patterns are the important characteristics of estuaries. Microbial communities are involved in mineralization of organic matter; therefore, I was interested in understanding the response of these sedimentary microbial communities to these regional and seasonal changes using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems .

Kranti: Everybody has someone in their life who inspires them to achieve something. Who is your inspiration in science?
Dr. Neetha: My PhD guide at NIO, Kochi is my inspiration in Science. She inspired me a lot! She encouraged me in various aspects of science and helped in boosting my confidence.

Kranti: Which methods and tools you use in your research?
Dr. Neetha: Microbial communities are involved in mineralization of organic matter in estuarine sediment. To understand the response of these microbial communities to various physiochemical and geochemical factors using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems. Phospholipids are mainly found in the cell membrane, not in storage lipids and have a rapid turnover in aquatic sediments. So it provides a measure of viable cellular biomass in an ecosystem.  Different physiological and functional groups of microorganisms in sediments were described using PLFA analysis.
The extracted PLFAs were analyzed using gas chromatography (Agilent 7890 Series, USA) with a cross-linked phenyl – methyl siloxane capillary column (25 m, 0.2 mm) and FID. Identification of the FAMEs was carried out by comparison of retention time and equivalent chain length with known standards like Eukary calibration mixture – 1201A (Eukary6 method, Version: 3.7) and MIDI peak identification software (MIDI Inc., Newark, DE).

Kranti: You are contributing to microbiology related services offered at NCMR Pune. What are those services ?
Dr. Neetha: I am in – charge for FAME analysis service and curator of Firmicutes at NCMR. Under FAME analysis, the bacterial (aerobic and anaerobic) or yeast samples are identified based on their cell membrane fatty acids. Also cell membrane fatty acids are analyzed for novel taxa along with their closely related type strains for publication.

Kranti: Are journals necessary in the age of internet? Don’t you think research should be done not just to publish a paper but also to have real life impacts?  
Dr. Neetha: We know that nowadays we can extract all the information we require via internet. But we cannot compare the beauty of reading a book or journal with internet. Yes, I totally agree that we should do research not only to publish a paper but also to have real life impacts.

Kranti: Being a woman in science, what are the challenges that you’ve faced?
Dr. Neetha: Being a woman in science, the major challenge I face is to manage family, children and their education along with my research work. Another challenge is to get time to spend for research along with my routine services and other commitments.

Kranti: How do you maintain the balance of your family and work-life?
Dr. Neetha: For that I should thank my husband and children for their co-operation and moral support throughout my career.

Kranti: What advice would you like to give to young women who want to pursue research?
Dr. Neetha: If you have an actual interest in science along with sincerity, dedication and hardworking nature, you will be able to succeed in your research career. As a woman, you should be able to manage your time and having patience is also equally important to succeed in your life.

Kranti: Would you share with us any memorable incident/moment of your research life?
Dr. Neetha: In the year 2000, I got an opportunity to participate in Cochin – Alleppey – Mangalore Cruise on board CRV Sagar Paschimi, under DOD, COMAPS Programme. It was a rare experience and golden memory in my research life.

Kranti: Most of the scientist’s children opt for career in science. Do you want your child to become a scientist too? 
Dr. Neetha: Yes, if they are showing real interest in science and research, definitely I will encourage him or her to opt for career in Science.

Plant growth promoting potential of a bacterial isolate from Tea garden in Assam

Tea is an economically important crop cultivated under warm and humid conditions. Assam is one of the largest tea-producing states in India. The tropical climatic condition gives the tea its unique malty taste but it also makes tea more prone to fungal diseases, which ultimately results in economic loss. Factors like insect/pathogen attack, drought, and heavy metals contribute to significant loss in tea production. Fungal diseases are significant contributors in reduced productivity of tea crops. Specifically, in the tea sector, estimated crop loss due to disease, pest, and weeds is reported to be around 85 million kg. Traditionally, chemical fertilizers, pesticides and insecticides are routinely used in tea gardens to tackle biotic stress factors. These chemicals are harmful for the ecosystem. The presence of pesticide residues in Assam Tea is a cause of great concern.

Plant Growth Promoting Rhizobacteria (PGPR) contributes in plant growth promotion activities, which eventually contributes to better farming. PGPRs colonize plant roots and benefit the plant system by solubilizing minerals such as phosphate, fixing Nitrogen in roots, producing phytohormones such as auxin; producing siderophore, iron scavenging molecules. They are also known to induce systemic resistance thereby developing robust mechanisms to resist biotic and abiotic stress.

Although PGPR associated with crops such as wheat, maize, rice, etc. have been widely studied, it is important to note that, despite Assam representing the largest tea producing states, the rhizosphere of tea has been poorly explored. Even in comparison with other crops, this commercial crop is highly ignored. Thus, it is important to characterize bacteria isolated from the tea rhizosphere and understand their functional potential for PGP traits, including biocontrol activity against pathogenic fungi.

Researchers from Assam University, NCMR-NCCS Pune and SPPU Pune conducted a study in which 23 distinct bacterial morphotypes were isolated from the tea garden of Assam, India. The isolates were screened for their plant growth promotion (PGP) and antifungal traits against three pathogenic fungi, namely Rhizoctonia solani, Corticium rolfsii, and Fomes lamaensis. Out of 23 isolates, 7 isolates showed potential in antifungal activities, amongt which, isolate A6 was found to have promising PGP and antifungal traits. Isolate A6 also exhibited biosurfactant production abilities. Biochemical and molecular characterization revealed its identity as Brevibacterium sediminis.

Biofilm – forming ability of fresh A6 culture was also assessed. Biofilm formation is considered as a prerequisite to colonize plant roots. Only after root colonization, the bacterium can perform the PGP activities. The finding of the study revealed that the isolate A6 forms moderately adherent biofilm. Biosurfactants now addressed as ‘green surfactants’ are well documented in the literature for plant growth promotion by their detrimental effect on pathogens. Hence, these biosurfactants and/or biosurfactant producing microbes are potential substitutes for the harsh chemical pesticides and insecticides being currently used in agriculture.

The study indicated isolate A6’s ability to exhibit PGP properties including the biocontrol activity and biosurfactant production and also to withstand the environmental stress such as its ability to grow and remain metabolically active in acidic pH. Also, the current findings need validation of reproducibility in tea plants. However, this study suggest and indicate that the bacterial isolate Brevibacterium sediminis A6 can be a potential PGPR candidate to be used in combination with other PGPR isolates for improving crop health and eventually the overall crop productivity.


Pseudomonas aeruginosa RTE4: A Tea Rhizobacterium with Potential for Plant Growth Promotion and Biosurfactant Production

In India, Assam is one of the largest producers of tea with high tea export rate. Tea plants are affected with fungal and bacterial diseases. Various chemicals like pesticides are used to combat these diseases which cause damage to plantation soils. Agricultural land in India has been depleting steadily over the years. The pesticides create pollution adding to the challenges of agriculture sector.

A study conducted by researchers from Assam university, SPPU and NCMR Pune explored the potential of native microbes at Rosekandy Tea estate in Assam and their secondary metabolites to check if they can replace the harsh chemical fertilizers. Based on earlier published research, Rhizosphere associated bacteria acts as Plant Growth Promoting Rhizobacteria (PGPR). PGPR benefits host plants by synthesizing phytohormone, solubilizing minerals in soil, fixing molecular nitrogen, controlling phytopathogens, producing antibiotics for disease suppression etc. Secondary metabolites including bio surfactant (BS) also referred as “Green Surfactant” is reported to improve soil quality, involved in PGP activity, and degrade/solubilize pesticides. Pseudomonas strains are one of the promising microbial systems which can be exploited for production of surface active agents. This study explored the BS producing abilities of rhizobacterium Pseudomonas aeruginosa RTE4 isolated from tea rhizosphere of Rosekandy Tea garden located in Cachar district of Assam.

Techniques like Colony morphology, Genome sequencing and phylogenetic analysis were used to identify Rhizobacterium RTE4. Antifungal attributes of the strain RTE4 were screened by identifying production of various hydrolytic enzymes like protease, cellulase and chitinase. The strain RTE4 showed strong antagonistic activity against both Tea plant pathogens C.invisium and F.solani . Inhibition in growth of X.campestris in a co-culture environment was indicative of antibacterial property of the strain RTE4. Pathogenic fungi and bacteria analyzed were susceptible to the rhamnolipid extract (BS) of RTE4 and to standard rhamnolipid as well. However, it is further essential to understand the mechanism by which BS portrays antagonism. Additionally, it is important to explore more microbes for rhamnolipid production considering the huge demanding global BS market. To meet the fungicidal efficiency of BS in fields, large scale BS production strategy needs to be explored so that BSs can be used as bio-fungicide.

Various physio-chemical properties of the purified BS were studied using techniques like Fourier-Transform Infra-Red Spectroscopy, Nuclear Magnetic Resonance Spectroscopy, and Liquid Chromatography-Mass Spectrometry etc. It demonstrated good surface wetting properties (parafilm and Teflon) with poor emulsifier properties. The anionic nature of RTE4 BS was confirmed using double diffusion method. Thin layer chromatography of the BS revealed its glycolipid nature. Antimicrobial activity of BS from RTE4 was tested against Tea pathogenic fungi (two) and bacterium (one) namely C. invisium, F. solani and X. campestris respectively. In control wells without addition of RTE4 BS or carbendazim heavy fungal growth was observed.

In order to replace the harmful chemical fertilizers with native rhizobacteria, it is important to understand the plant growth promoting potential of the microbes in the rhizosphere. The various metabolites secreted by rhizobacteria that can help growth and health of the host plant. Microbial BS is advantageous over chemical surfactants in various properties, both physical and chemical. The phenomenon of bacterial quorum sensing (QS) is knows to regulate the PGPR traits. Therefore, further exploring QS potential of the isolate will help us to enhance our knowledge on the role of QS in BS production in the isolate RTE4.

Several nutrients are essential for plant growth and development. Phosphate is second most important macronutrient required by plants after nitrogen. Unlike nitrogen, phosphate cannot be made biologically available from the atmosphere, hence lays the importance of phosphate solubilizing bacteria which help plants to solubilize inorganic phosphate present in soil. The metabolic pathway involving glucose for microbial production of rhamnolipid has been researched well enough. Glucose is a preferable carbon source because it can be converted to precursor molecules required for rhamnolipid synthesis. In this study, it was also found that among all other carbon sources tested, glucose proved to be the best one for the growth and production of BS from P. aeruginosa RTE4. The thermal and pH stability of BS was studied because it is important for its commercial application. BS from RTE4 also maintained its stability at varying temperature conditions from −20 to 121◦C making it efficient for commercial applications.

In conclusion, efficacy of a microbe to be exploited as bio-fertilizers first needs an understanding of the mechanisms by which it shows plant beneficial traits. Many bacteria which prove to be beneficial for plants are controversial for being pathogenic to humans. Therefore, digging out the targeted secondary metabolites from such plant-native microbes eliminates the fear of its danger to humans. P. aeruginosa is one such controversial species which is widely found in various plant rhizospheres and yet claimed to be a PGP. The study conducted show that the strain RTE4 exhibits multiple PGPR attributes along with production of BS molecules having huge potential for their applications in agriculture as bio-fungicide.


Identification of a strain related to phytoplasma infection in marigold in India

Phytoplasma is a group of extremely small bacteria. They don’t have a cell wall and any particular shape. They are bacterial parasites of plants and insects. Marigold is an important floricultural crop which has been reported as phytoplasma host from many countries like Mexico, India and Hungary. The state of Karnataka is one among the leading producer of Marigold flowers in India. Marigold is the most widely cultivated commercial flower of Karnataka, it has a significant role in the national and international flower trade. Phyllody and associated symptoms of phytoplasma infection have a direct negative impact on the quality and market value of marigold flowers.

Typical disease symptoms on marigold plants, from the left: healthy marigold plant with normal inflorescence, and with virescence and phyllody symptoms.

Researchers conducted field surveys in the marigold growing areas of Karnataka from April 2016 to July 2018 to assess the presence of phytoplasma associated symptoms in the crop. A total of ten symptomatic and two asymptomatic samples were collected and the percentage of disease incidence was determined by taking into account the number of diseased plants for every 100 plants evaluated. DNA extraction was done from the leaves of symptomatic and asymptomatic plants. Characterization of phytoplasma was done using direct and semi-nested PCR assays. The virtual RFLP patterns were generated and compared with the reference phytoplasma strains of different 16Sr groups. The assembled 16Sr sequences and the sequences retrieved from the GenBank were aligned and
a phylogenetic tree was constructed.

The symptoms of phyllody, yellowing, discoloration of florets, stunted growth, purpling and crinkled leaves were observed on marigold plants. The virtual RFLP patterns resulted identical with the one of papaya yellow crinkle phytoplasma, ‘Candidatus Phytoplasma australasia’ classified in the 16SrII group, subgroup D with the similarity coefficient of 1.00. In this study, the symptomatic samples of marigold collected from different fields resulted infected with a ‘Ca. P. australasia’-related strain, which has not been reported in India in this plant species. This study is the first report of 16SrII phytoplasmas associated with marigold phyllody from the Karnataka state.


A threat to Sandalwood cultivation in Marayoor Sandalwood Reserve through phytoplasma infections

Santalum album, commonly known as Indian Sandalwood is a semi parasitic plant with fragrant wood and is one of the most valuable trees across the globe. The wood and roots contain ‘sandal oil’ which is valued for use in perfumes, incense, cosmetics, soaps, and medicines. The bark contains tannin, which is used for dye production. The states of Karnataka, Tamil Nadu and Kerala are recognized for natural populations of sandalwood contributing 90% of its distribution in India.

Sandalwood Spike Disease (SSD) is the most destructive disease of sandalwood and one of the major cause for decline in sandalwood production. SSD is known to be associated with the presence of aster yellow (‘Candidatus phytoplasma asteris’ 16SrI-B) phytoplasma. Phytoplasma is a group of extremely small bacteria. They don’t have a cell wall and any particular shape. They are bacterial parasites of plants and insects. SSD is identified based on presence of chlorosis, reduction in leaf size, and shortened internodes, causing leaves to become crowded on twigs with a bushy appearance and stems standout stiffly with spike-like appearance.

Researchers collected the symptomatic sandalwood samples from the Marayoor sandalwood reserve in Kerala state and and they confirmed the presence of 16SrI-B phytoplasma, however 16SrXI-B group phytoplasmas were also identified. These phytoplasmas were present in single or in mixed infection in the collected sandalwood, sugarcane and Indian gooseberry samples from this area. This study is the first report of 16SrXI-B phytoplasma presence in SSD plants.

(A) A diseased and symptomatic sandalwood branch showing typical little leaf and spike symptoms,
(B) A dead sandalwood plant, due to spike disease

The study reconfirmed the earlier observation of the presence of aster yellows phytoplasmas in plants with SSD disease, but those reports were from Chamundi Hill areas of Karnataka state. There is a great fear of complete loss of sandalwood cultivation from Marayoor Sandalwood Reserve region, which is the only naturalised and largest reservoir in the world. The spread of SSD to the neighbouring sandalwood reserves of Marayoor will cause drastic damage. This study reports the first 16SrXI phytoplasmas detection in plants with SSD disease.


Phytoplasma associated with witches’ broom disease of a weed plant

(A) Healthy C. bonplandianum plant twigs with inflorescences
(B and C) Symptomatic plants showing witches’ broom, yellowing and little leaf symptoms

Croton bonplandianum is an exotic weed which is wide spreaded in South America and in many countries of the Indian subcontinent. C.bonplandianum is commonly known as three-leaved caper, “Ban Tulsi”, or “Kala Bhangra”. In India, this weed primarily grows in barren lands with sandy clay soil, and is commonly found in the fields of paddy, sugarcane, vegetable and fields of pulse crops. This weed is used in traditional medicine. Other species of the genus Croton are used as ornamentals, indoor plants and are potential biofuel source.

Phytoplasma is a group of extremely small bacteria. They don’t have a cell wall and any particular shape. Phytoplasma was first identified by a Japanese scientist Yoji Doi as ‘mycoplasma-like-organisms’ in 1967. They are bacterial parasites of plants and insects.  The study was conducted by researchers at NCMR pune in order to understand whether there is any phytoplasma association with the witches’ broom disease in C.bonplandianum plant. In this study, four symptomatic plants showing typical witches’ broom, little leaf, yellowing, virescence and phyllody symptoms along with healthy samples were collected from Baramati, India. To confirm the phytoplasma presence, leaves and floral whorls were used for total DNA extraction and further experiments were performed.

The study reported 16SrII group phytoplasma association with witches’ broom disease of C. bonplandianum. This is the first identification of a ‘Candidatus Phytoplasma australasia’-related strains in C. bonplandianum showing witches’ broom symptoms. Studying phytoplasma infection in these weeds becomes highly significant in agriculture as they cause devastating yield losses in diverse crops worldwide. Early identification of phytoplasmas infecting weed is crucial to verify the possibility of the spread of phytoplasmas diseases to commercial crops.


Root-associated microbiome of oxalogenic plant reveals distinct bacterial diversity

Colocasia esculenta (wikimedia)

Colocasia esculenta (Linn) also known as Taro is a tropical plant primarily grown as a vegetable food for its edible corm, and secondarily as a leaf vegetable. Colocasia esculenta grows relatively low to the ground and is a tuberous plant in the family Araceae. Colocasia esculenta has been documented to have oxalogenic properties. Oxalate is found in different environments such as soil and gastrointestinal tracts. Oxalate metabolizing bacteria also known as oxalotrophic bacteria can metabolize oxalate for carbon and energy source. Some plants produce the oxalate crystals as a defense against the herbivory. Oxalotrophy is involved in root colonization by plant-associated bacterial species that may have a positive role in plant growth.

A collaborative study was conducted by researchers at NCMR-NCCS Pune, Yenepoya Research Centre Mangalore, SPPU Pune, University of Nevada, Las Vegas, USA and Zeal College of Engineering and Research Narhe, Pune with the aim of understanding the rhizospheric microbial communities in an oxalogenic plant with the prospects of recognizing possible bacterial species for their capability to metabolize oxalates.

Researchers collected naturally growing Colocasia esculenta (Local name: Arum) plants from a botanical garden near National Centre for Cell Science, Pune. The plant roots and surrounded area were selected and sampled as non-rhizospheric (NS), rhizospheric (S) and rhizoplane (P) soil fractions.  DNA isolation and further 16S rRNA gene amplification and sequencing were done for the samples. Taxonomic assignments and statistical analysis was performed.

Total 852 sequences were obtained from the three root compartments. Out of these, 311 corresponded to rhizosphere, 250 to rhizoplane and 291 were from non-rhizospheric soil. Bacteria belonging to phylum Proteobacteria were recorded relatively higher across all samples. Firmicutes in rhizosphere (S) soil, Actinobacteria in the non-rhizospheric (NS) soil whereas, Bacteroidetes (12%) in the rhizoplane (P) soil were found to the second most abundant groups. Flavobacteriaceae, Enterobacteriaceae, Moraxellaceae and Pseudomonadaceae were the major contributors in the rhizoplane microbial community assemblage. Paenibacillaceae was the major contributor to the rhizospheric microbial community. There were no species belonging to Firmicutes that were shared by nonrhizospheric (NS) and rhizoplane (P) soil samples. However, 6 bacterial species were shared by non-rhizospheric and rhizospheric soil samples. 6 bacterial species were exclusively present in the rhizoplane compartment which constituted of species belonging to genus Exiguobacterium, Paenibacillus, and Solibacillus.

In this study, the results indicate a clear distinction in the microbial community diversity at the phylum level. The complete absence of members of the phyla Cyanobacteria, Gemmatemonadetes and Planctomycetes from the rhizosphere and rhizoplane microbial population supports the role of root exudates in significantly influencing and designing the microbiome. Since Colocasia esculenta is an oxalogenic plant and has been known to release oxalates in the root exudates, the predominance of Proteobacteria in the rhizosphere and rhizoplane microbial communities indicates the oxalotrophic activity which might be a major functional trait of the communities associated. The study concluded that, the rhizoplane has a distinctive composition of microbial partners as compared to the rhizosphere and bulk soil communities in Colocasia esculenta.


Analysis of SARS-CoV-2 genomes from western India reveals unique linked mutations

Transmission electron micrograph of SARS-CoV-2 (Wikipedia)

COVID-19 is caused by the strain of corona virus named Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), belonging to the category of betacoronaviruses. The virus mainly causes respiratory illness, varying in severity for different individuals. The COVID-19 pandemic is affecting the whole world. India is one among the worst hit nation by the COVID-19 pandemic. The western part of India is badly affected by the COVID-19 pandemic, the Maharashtra state is a major hotspot for this disease, having around 1/5th of total reported infections in India.

A collaborative research conducted by researchers from NCCS Pune, B. J. Government Medical College, Pune and Armed Forces Medical College Pune present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India. In this study, researchers have investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in three different regions of Maharashtra, the western state in India. Total 90 genomes were sequenced. The analysis revealed three unique linked mutations which are common in most of the sequences studied. These may act as molecular markers to track the spread of the SARS-CoV-2 virus to different areas.

Nasopharyngeal/throat swabs of suspected COVID-19 patients were collected, samples confirmed with SARS-CoV-2 infection were used for the study. The age of the patients selected in the present study ranged from 2-78, with 80% patients were in the age range of 30-60 years. COVID-19 patient samples with a particular range Ct value for E gene were selected for the genome sequencing. Fast qc tool and BWA (Burrows-Wheeler Aligner) were used for data analysis. Neighbor joining method was used for phylogenomic analysis. Structural and bioinformatics analysis of SARS-CoV-2 variants was performed and comparative study among the Indian samples was also done. The observed mutation pattern was further analyzed to check any relationship with gender, age, and symptoms.

Phylognetic analysis of the genomes revealed that mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples, indicating a newly emerging pattern of linked mutations. The Satara district viral strains showed mutations primarily at the 3´ end of the genome, while Nashik district viral strains displayed mutations at the 5´ end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients was performed. The analysis showed mutations were prevalent in symptomatic cases, and were more prominent in females. These three mutations were present in more than 30% studied samples of age group 10-25. Interestingly, these mutations were not detected in the higher age group of 61-80.

Study of region-wise mutation pattern among the viral sequences indicated that, a specific pattern of mutation was prevalent in all districts. The relationship of mutation pattern with age, gender and symptoms was studied. A distinct pattern was observed in age-wise distribution, some of the mutations were prevalent in the age group of 10-25. The proportion of three mutations C313T, C5700A, G28881A were found relatively higher (~80%) in symptomatic patient samples as compared to asymptomatic (40-50%). Also, the mutation C241T was found in 90% of all the sequences and is located in the 5′ UTR region and found predominantly in severely affected patients. However, the role of this mutation has not been studied yet.

The comparative study indicated that, distinct sub-clones of virus were prevalent in different parts of India at the same time period. The type 19A clade virus was predominant in Delhi (Northern part) whereas in Maharashtra (western part) 20A, 20B clade virus was dominant in April-May 2020. While in Telangana (southern part), 19A clade was dominant in April, and it shifted completely to 20A and 20B in May 2020. Because of lockdown, factors contributing to transmission of SARS-CoV-2 virus was restricted. The researchers are assertive about prevalence of a specific viral variant in a region could be attributed to human host susceptibility for specific viral variants. This susceptibility seems to be based on mutations prevalent in the viral variants in that region.