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.