Arvind K. Misra

Taxonomic resolution of actinorhizal Myrica species from Meghalaya (India) through nuclear rDNA sequence analyses

Three different morphological variants of genus Myrica L. (Myricaceae) are found in Meghalaya, India. There is a dispute regarding their classification. Some authors treat them as two separate species (Myrica nagi Thunb. and Myrica esculenta Buch.-Ham. ex D. Don) whereas others feel that M. nagi and M. esculenta are synonyms. In an effort to resolve this dispute, we investigated the nuclear rRNA genes segment of DNA of these variants utilising the amplicon restriction patterns, nucleotide sequences and secondary folding of the 5.8S rRNA. It is proposed that M. nagi and M. esculenta be treated as two separate species.

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region ofrrn operon ofFrankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS 1 region of the nuclearrrn operon ofAlnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

Molecular diversity of Frankia from root nodules of Hippophae salicifolia D.Don found in Sikkim

Molecular diversity of Frankia was assessed directly from the root nodules of Hippophae salicifolia naturally occurring in North Sikkim. Amplicon restriction patterns (ARPs) were developed by digesting 16S-ITS-23S amplicons with RsaI. Three ARPs were detected, showing diversity among strains of Frankia that nodulate Hippophae. This was confirmed by sequencing one amplicon each for the three ARPs. Therefore, ARP can be used as a tool for screening amplicons for nucleotide sequencing.

Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts

Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since.

Phytometabolomic fingerprinting of selected actinorhizal fruits popularly consumed in North-East India

The biodiversity hub at north-eastern region of India contains actinorhizal plants like Elaeagnus pyriformis, Myrica nagi and Myrica esculenta which serve as functional food for the natives. Apart from having immense nutritional value, fruits of these plants have been utilized in traditional medicine and have displayed antioxidant behavior as observed from our previous findings. Therefore, the aim of the present study was to investigate the phytochemical fingerprints of the fruit juices of some actinorhizal plants using GC-MS method and correlate the data with the therapeutic value of the fresh fruit juice. The results established the abundance of several chemical species with potential medicinal properties such as anti-diabetic, anti-cancer, anti-inflammatory, hepatoprotective activities. However, further studies are needed to isolate and identify the specific phyto-compounds involved in managing various diseases.

Immobilized Frankia spores remained viable on dry storage and on restoration to medium regenerated active colonies

Spores of Frankia strain ACN1AG, immobilized in calcium alginate beads, germinated to produce colonies that increased in protein content and showed nitrogenase activity. Air dried immobilized spores remained viable for at least 15 days in dry condition, making the storage and transport of Frankia strains easy. This also opens the possibility of using beaded spores as inocula.

Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

Frankia and Actinorhizal Symbiosis

The actinobacterial member Frankia has a special significance as nitrogen fixing partner in symbiosis with some non-leguminous plants. Frankia seems to have entered into symbiotic association with many diverse dicotyledonous plants belonging to eight families independently several times during the course of evolution of this important symbiosis. Since this association confers a special ability to colonize soils low in nitrogen, actinorhizal plants are able to act as pioneers in the regeneration of Jhoom fallows, land slide affected areas, mine spoils, etc. They are also useful as wind breaks and as sand dune stabilizers. The accessibility of the complete genome sequences for three Frankia strains has provided new evidences pertaining to its evolution and structure, interactions between actinorhizal plants and their diversity. The establishment of the structure of NifH proteins from Frankia provided significant insights into the structure-function relationships. Analysis of the nif genes and whole genomes of Frankia using nucleotide triplet based phylogeny highlighted the roles of lateral gene transfer and gene duplications.