Phytoplasma: A tiny microbe affecting multiple plant species

By Kranti Karande

Phytoplasma is a group of extremely small bacteria (mollicutes). They don’t have a cell wall and any particular shape (pleomorphic). Phytoplasma was first identified by a Japanese scientist Yoji Doi as ‘mycoplasma-like-organisms’ in 1967. They are bacterial parasites of plants and insects. Phytoplasmas reside in plant’s phloem tissue while insects serve as vectors for the transmission of infection from plant to plant. Once disease caused by phytoplasma is established, entire fields of crops might be wiped out. Indian sandalwood production has been drastically reduced due to phytoplasma infection during the last few decades.

The major characteristic symptom of phytoplasma-caused disease is abnormal growth of the floral organ,green flowers development (phyllody) and numerous growth of tree branches which appears like ‘Witches’ boom’ and reduction in leaf size called ‘little leaf’. Phytoplasma infection also causes curling and yellowing of the leaves (virescence). Disease caused by phytoplasma is called Sandalwood Spike Disease (SSD) in sandalwood plants, as leaves show growth in ‘spike-like’ structure. In few species of plants, such as onion and bamboo, the symptoms of witches’ broom and shortening of plant height (stunting) are observed due to phytoplasma infection.

Some plant pathogens produce virulence factors (effectors) that modulate or interfere with normal host processes for the pathogen’s benefit. Phytoplasma is reported to express secretory proteins including SAP proteins. The major obstacle limiting research in phytoplasma disease is that the organism is difficult to cultivate in vitro (laboratory cultivation).

Dr. Amit Yadav’s research group at NCMR, NCCS Pune is interested in understanding these pathogenic microbes which cause disease in over a thousand species of plants and affect agricultural productivity around the world. They’re interested in understanding genetic factors related to pathogenicity and phytoplasma transmission. They study phytoplasma taxonomy and the role of insect gut micro biome in vectoring ability of the insects.

It’s important to study the genome of this organism to understand how this tiny microbe causes infection in plants and gets transmitted through insect vectors. Phytoplasma DNA is difficult to isolate and then sequence it further, as researchers have not yet been active in this organism’s laboratory cultivation. Dr. Yadav’s research group is currently working on 40-45 symptomatic plants associated with phytoplasma infection. Recently, the group was successful in isolating and sequencing sugarcane phytoplasma. Dr. Yadav’s group is interested in studying other phytoplasma species that are prevalent in India; mainly associated with diseases in economically important pulse crops, sandalwood and weed species; and will be the first to publish phytoplasma genome sequence from India.

There is no cure yet available for this infection. Phytoplasmas are transmitted from plants to plants via the insect vectors. Weeds are also known to be secondary phytoplasma carriers. The weeds around the field are not eradicated by farmers, insects introduce phytoplasma to these weeds and then again to the new crop transmitting phytoplasma infection. Dr. Yadav’s group is also interested in studying the insects’ vector families and mechanism involved in the transmission of infection with Phytoplasma.

Some researchers are trying to understand if phytoplasma are beneficial for the insect vectors transmitting phytoplasma, and why some insects act as transmitters and some not.

Phytoplasmas have one of the smallest genomes as compared to other common bacteria. They completely rely on their host for nutrient supply, growth, spread and reproduction. “How organism with such a small genome affects over 1000 plant species is a curious question that drives me in this study,” said Kiran Kirdat, researcher from Dr. Yadav’s group. She also commented that “Dr. Yadav has been a great motivator for me”.

Dr. Yadav’s group has reported novel species of phytoplasma from various economically important plants like from Mango, Sandalwood, Sugarcane, Tamarind etc. Along with taxonomy study their research focus is also on understanding role of insect gut microbiome in the vectoring ability of these insects. They are interested in understanding the diversity of microbiota in the gut of insect vectors. The Wikipedia page of ‘Sugarcane Grassy Shoot Disease’ has been created by Dr Yadav and he has contributed significantly in other phytoplasma related article on Wikipedia.

Dr. Yadav said that, “People are more aware of phytoplasma associated diseases than a decade ago, when I started working on this notorious bacterium. However, much needs to be done further. Many crops like Sugarcane, Sesame, Sandalwood, coconut in India are facing great challenge to sustain due to phytoplasma infection. We are working on two different aspect to study phytoplasma; one is to understand how this pathogen spread the disease from one plant to other through insect vector and other is to study the genome of this organism to find out disease biology. We are also working on development of reliable and robust detection of phytoplasmas to help on field workers.”

Phytoplasma affects many economically important plant species and thus is a major threat to agriculture economy. Understanding the epidemiology of the phytoplasma diseases, pathogen and genomics of this tiny microorganism will help to cure the infection in future.  

MALDI-TOF Mass Spectrophotometry (MS): Emerging technique in Microbial Ecology

-By Kranti Karande

Microorganisms have been identified by their biochemical properties and using techniques like 16S rRNA sequencing for more than a century. Recently, MALDI-TOF Mass Spectrometry (MS) has become a key technique for microbial identification due to its rapid and reliable performance.

MALDI-TOF MS facility at National Centre for Microbial Resource, National Centre for Cell Science Pune

Mass Spectrometry is an important tool for the detection of atoms or molecules in a chemical complex. A mass spectrometer works by turning atoms into ions. Ions get separated when they are passed through electric field and magnetic field respectively. A spectrum is generated determining types of atoms present in the sample.

MALDI-TOF MS technique has a variety of applications in biology including intact mass determination, peptide mas fingerprinting, MALDI imaging. This method is widely used for the identification of DNA and proteins, but recent advances in this technique have incorporated use in microbiology for pathogen detection, which is contributing immensely to research in microbiology.

Dr. Praveen Rahi from NCMR-NCCS Pune and Dr. Parag Vaishampayan from the California Institute of Technology, USA, recently published an editorial detailing the recent advancements and challenges in MALDI-TOF MS for Microbial Ecology applications.

This technique is in the early stage of development for the direct identification of microorganisms in positive blood cultures, detection of drug resistance factors and for bacterial function assessment. Recently, researchers identified Brucella infection from positive blood culture using this technique which is a great breakthrough in MALDI-TOF MS application. Researchers are also trying to develop this technique for studying antimicrobial resistance in bacteria and fungi.

In one of the recent publications, data from MALDI-TOF spectra of intact protein and specialized metabolite spectra from bacterial cells grown on agar were combined to study the link between microbial identities and their potential environmental functions.

This technique is becoming a fast and effective method for filtering out same species strains and genetically identical clones. Until now, identification of same species strains was done with the help of techniques like dendrograms. Now MALDI-TOF MS is used for identifying identical strains as it provides accurate results with rapid analysis.

With the help of MALDI-TOF MS technique medical researchers revealed a potential clonal route of transmission of Enterobacter spp. This research is a significant source of information on the spread of Enterobacter, an evolving pathogen for its ability to acquire antimicrobial resistance factors and the role of MALDI-TOF MS in microbiological surveillance of diseases.

Dr. Praveen Rahi
NCMR, NCCS Pune

While discussing about MALDI-TOF MS applications with Dr. Praveen Rahi, he commented that, “MALDI-TOF MS will be ‘new microscope’ for microbiologists in the era of ‘culturomics’, which include large-scale cultivation of microorganisms and require high-throughput identification tools. Right now the commercial MALDI-TOF MS spectral databases primarily include profiles of microorganisms of clinical relevance, and researchers across the globe are making efforts to generate spectral profiles for microbes according to their research interests. However, most of these efforts are being made in isolations, which might lead to erroneous database entries. There is urgent need to develop universal SOPs for MALDI-TOF MS profile generation, and subsequent curation and validation of newly generated database profiles.”

Though this technique has emerged as a key player in microbial identification and microbial ecology research, but there are many fine tuning steps required to improve this technique.

One study noted that only 35% of species level identifications done using MALDI Biotyper correlated with those detected by 16S rRNA gene sequences, suggesting a poor species level detection by this technique. Insufficient coverage of bacterial species in the repositories was proposed as the primary reason for this difference. The lack of a public repository to send new spectral references created by researchers further worsens this issue. The major challenge is to find the solution for same.

During the identification of the collection of microorganisms isolated from cell phone surfaces, researchers observed that the efficiency of MALDI-TOF MS spectrum filtering can be achieved by improving the methods of sample processing and enriching the spectral database leading to better and highly reliable results for MALDI-TOF MS microbial identification.

If we find feasible solutions for the challenges in this technique, this technique has the potential to make an immense contribution to the field of microbial ecology.

NCMR-NCCS Pune provide services for microbial identification using MALDI-TOF MS.

Reference: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02954/full

Scientists report that delay in sample processing affect the cell viability

Dr. Avinash Sharma
NCMR, NCCS Pune
Wellcome Trust DBT
Indian Alliance Fellow

-By Kranti Karande

Sambhar Lake, located in the Rajasthan district, is the largest inland salt lake in India. It is a Ramsar site, a saline wetland of international importance because of the spectacular diversity of birds it attracts. The salt production from the lake is approximately 0.2 million tons per annum. Apart from this it is also considered as a hypersaline ecosystem because of high salinity which provides a great opportunity for microbial ecologists to understand halophiles. Over the last many years there are very few reports on the microbial diversity of halophiles, in particular archaea from Sambhar Lake.

Dr. Avinash Sharma’s group at NCMR-NCCS Pune cultivated diverse group of bacteria and archaea from Sambhar Lake. Archaea are very difficult to culture and not many research groups are working on this domain of life. Looking at the percentage of description of novel taxa belonging to bacteria and archaea, one can easily make out how poorly archaea are being studied.  13 genera were identified as archaeal while 12 genera belonging to bacteria were isolated in the current study. The authors also claim that there are 13 novel strains of archaea and bacteria isolated during study based on 16S rRNA gene sequencing and currently the group is working on genome analysis which is mandatory for the description of a novel taxa.   

        The reason behind capturing such a vast diversity was because of the multiple cultivation approaches used in the study.  The cultivation of microorganisms was done using two used approaches: onsite enrichment and sediment dilution in the laboratory. In the first method of cultivation, soil samples were inoculated in different growth media onsite. The same media and salt concentrations were also used for isolation in the second approach. That is same growth conditions were used in both approaches. Interestingly besides common found genera in both cultivation methods: AlteribacillusHalobacillusHalorubrumLentibacillusNatronorubrum
Piscibacillus and Thalassobacillus were found only in onsite cultivation whereas only three genera AliidiomarinaNatrinema and Natronolimnobius were found in laboratory processing.

Dr. Avinash Sharma said “Although both the approaches used for isolation are classical, but they provide better understanding to device sampling strategies. From our results we can conclude that it is important to use multiple cultivation approaches as these approaches help capturing vast group of organisms. Imagine if we would not have done the onsite enrichment we would have lost majority of the organisms we isolated in this study. We have also observed in the study how delay in sample processing effect the cell viability, although it is not possible to isolate each and every organism residing in the ecosystem but immediate sample processing is recommended to isolate majority of the organisms before they lose their viability”.

   The microorganisms isolated in this study are known for the production of bio technologically important compounds like halocines, bacteriorhodopsins, exopolymer, hydrolytic enzymes and possessing the anti-proliferative, anti-oxidant and anti-hemolytic activity.

Conservation of such unique organisms at NCMR-NCCS enrich the culture collection and provide better resources for future studies/research.

Reference: https://www.ncbi.nlm.nih.gov/pubmed/31912221

A new bacterial species described from the salt water lake in India

-By Kranti Karande

A bacterial strain ( MEB193T )was isolated from the Lonar lake which is a notified national Geo-heritage monument situated in Buldhana district of Maharashtra, India .

As researchers collected the sample water, the water had a pH of 9.8 and a temperature of 28ºC. This bacterial strain represented as MEB193T was isolated on modified sea water agar.The strain was described as rod-shaped, motile and non-spore forming.

Dr. Amaraja Joshi, NCMR Pune
Dr. Amaraja Joshi NCMR, NCCS Pune

Dr. Amaraja Joshi’s group at NCMR, with Dr. Yogesh Shouche identified the taxonomy of this strain.

Based on draft genome sequence on the Illumina MiSeq platform, 16S rRNA gene sequencing and observable characteristics , they found that the strain MEB193T represents a new species of the genus Nitrincola . The strain name is proposed as Nitrincola tapanii sp. nov. , in the honour of Dr. Tapan Chakrabarti for his enormous contribution in the field of microbial taxonomy and systematics. Five species of the genus Nitrinicola were described before this research. In conclusion, a new species surviving in an alkaline environment was described by researchers.

Reference: https://www.ncbi.nlm.nih.gov/pubmed/31751193