Rakesh Pathak

Assessment of Genetic Diversity in Trigonella foenum-graecum Based on Nuclear Ribosomal DNA, Internal Transcribed Spacer and RAPD Analysis

Fenugreek (Trigonella foenum-graecum) is receiving global attention due to rare medicinal properties of significance to human health. Gene banks possess scanty germplasm and very little background information regarding its genetic variability that has hampered its improvement. We investigated the extent of variability among 17 Indian varieties of fenugreek using phenotypic and genetic markers. Multilocus genotyping by ten random amplified polymorphic DNA (RAPD) primers detected an average of intraspecific variations amounting to 64.7% polymorphism in banding patterns. Analysis of molecular variance indicated that a greater proportion of total genetic variation exists within population (91%) rather than among populations. Higher values of Nei’s gene diversity (h) and Shannon Information Index (i) and genetic distance analysis validate higher genetic diversity among Indian fenugreek varieties. SNPs at 14 sites of rDNA region revealed further lineages of distinct varieties with main RAPD clusters. The representative sequences of each subgroup and all distinct varieties have been submitted to NCBI database and assigned Gen Accession numbers HM 176640–176649. The measures of relative genetic distances among varieties of fenugreek did not completely correlate with the geographical distances of places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among fenugreek varieties studied. Eventually, the knowledge of their genetic relationships, DNA bar coding and phylogenies might contribute for the designing of intraspecific crosses between cultivars of this fenugreek collection with potential interest in seed spices breeding programme.

Molecular characterization of Vigna radiata (L.) Wilczek genotypes based on nuclear ribosomal DNA and RAPD polymorphism

Mungbean germplasm characterization, evaluation and improvement are fundamentally based on morpho-agronomic traits. The lack of break-through in mungbean production has been due to non-availability of genetic variability for high yield potential. Forty-four genotypes of mungbean [Vigna radiata (L.)Wilczek] were subjected to random amplified polymorphic DNA (RAPD) analysis to assess the genetic diversity and relationships among the genotypes. Multilocus genotyping by twelve RAPD primers generated 166 markers and detected an average of intraspecific variation amounting to 82% polymorphism in banding patterns. Dendrogram obtained from cluster analysis delineated all the 44 genotypes into six clusters. Higher values of Nei’s gene diversity (h) and Shannon information index (i) and genetic distance analysis validate existence of wide genetic diversity among mungbean genotypes tested. Besides internal transcribed spacer (ITS) length variations, single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) were detected at number of sites in nuclear rDNA region and the sequences of representatives of each sub-cluster and all distinct genotypes have been submitted to NCBI database and assigned Gen accession numbers HQ 148136-148147. Multiple sequence alignment revealed further lineages of distinct genotypes with main RAPD clusters. The measures of relative genetic distances among the genotypes of mungbean did not completely correlate the geographical places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among mungbean genotypes studied. RMG-62, RMG-976, and NDM-56 have been identified as potential source of parents for crop improvement. RAPD primers, OPA-9 and OPA-2 as polymorphic genetic markers and number of pods/plant and number of seeds/plant as dependable phenotypic markers have been identified for improving yield potentials. This genetic diversity will be of significance in developing intraspecific crosses in mungbean crop improvement programme.

Biodiversity in wood-decay macro-fungi associated with declining arid zone trees of India as revealed by nuclear rDNA analysis

Wood-decay macro-fungi have been reported to be a major cause of decline of arid zone trees in the genera Acacia, Prosopis, Ziziphus and Azadirachta; the identity of these fungi, however, has not been confirmed in the state of Rajasthan in northwestern India. Direct sequencing of PCR amplified ITS regions of rDNA facilitated molecular identification of 26 isolates into species in eight genera (Ganoderma, Inonotus, Phellinus, Ceriporia, Schizophyllum, Phanerochaete, Pleurotus, Leucoagaricus) and one incompletely characterized Basidiomycota species. In addition to intra-specific phenotypic variations in basidiocarp morphology, Single Nucleotide Polymorphisms (SNPs) were recorded among isolates of P. pulmonarius, S. commune, G. lucidum and Ganoderma species. Both SNPs and insertions/deletions (INDELS) were recorded amongst Inonotus species. A phylogram based on multiple sequence alignment delineated all isolates into separate intra-specific subgroups along with their GenBank reference sequences with high bootstrap values. Phellinus repandus, L. meleagris, C. lacerata and three Inonotus species (I. rickii, I. patouillardii, and I. porrectus) reported herein are new fungal records from India.

Genetic diversity of Indian jujube cultivars using SCoT, ISSR, and rDNA markers

Genetic variation and relationships among 37 cultivars of Ziziphus mauritiana (Lamk.) native of India were analyzed using start codon targeted (SCoT), inter-simple sequence repeats (ISSR), and ribosomal DNA (rDNA) markers. High level of polymorphism among SCoT (61.6%) and ISSR (61%) primers with higher PIC values ranging from 63.1 to 90.4% of SCoT and 47.3 to 88.8% of ISSR primers was recorded. SCoT and ISSR dendrograms revealed similarity coefficients ranging from 0.80 to 0.92 and 0.79 to 0.96, respectively, and clearly delineated all the cultivars of Z. mauritiana into well-supported distinct clusters. Greater Gst signifies higher amount of differentiation observed over multiple loci among seven Z. mauritiana populations. On the other hand, higher gene flow demonstrating a very high migration rate between Z. mauritiana populations indicated higher rates of transfer of alleles or genes from one population to another. The genetic diversity of population 1 (Rajasthan) was the richest among all the seven populations. The largest genetic distance was measured between Maharashtra and West Bengal and the least between Rajasthan and Punjab cultivars. Most of the genetic diversity exists within population rather than among populations. Substantial variation in the ITS-1 region signifies its phylogenetic utility specifically in assessing genetic diversity in Z. mauritiana. The clustering patterns using three molecular marker systems vis-à-vis place of origin exhibited no consistency in grouping of Z. mauritiana cultivars as cultivars from the same place of origin were genetically cataloged into different SCoT, ISSR, and ITS phylogram clusters indicating wide genetic diversity and distribution across agro-climatic zones validating the robustness of marker systems tested.

Seed Priming-Mediated Induced Disease Resistance in Arid Zone Plants

Priming of seed provokes plants to activate defence responses more quickly and effectively against phytopathogens without alternating plant growth and has the potential to emerge as a strategic tool for modern plant protection. Seed priming is an attractive, simple and cost-effective strategy that induces systemic resistance to control the plant diseases. Seed primed through microorganisms reflects biochemical/physiological changes leading to the synthesis of proteins and chemicals involved in induced systemic resistance and increases the efficacy of the plant against several pathogens. The present chapter summarizes the current knowledge of the seed priming and its relevance for plant protection with special reference to bio-priming.

Plant Growth-Promoting Rhizobacteria-Mediated Acquired Systemic Resistance in Plants Against Pests and Diseases

Plant growth-promoting rhizobacteria (PGPR) are indispensable part of rhizosphere microbiota that grow in association with the host plants and stimulate the plant growth. PGPR microcosm establishes in soil ecosystem because of its adaptability in varied environments with faster growth rate and biochemical versatility. In recent years researches have emphasised the key role of PGPR in improving nutrition and productivity of important crops with therapeutic and industrial significance. Hence, therefore, the present chapter highlights PGPR-mediated acquired systemic resistance against phytopathogens and insect pests involving mechanisms of action. Field applications of PGPR-mediated results reflect their substantial role in inducing systemic resistance in crop plants.

Cultivation, Conservation and Medicinal Significance of Macrofungi

Mushrooms are saprophytic macrofungi. Due to differences in sexuality patterns, mostly diploid pure mycelia cultures are raised as tissue culture on suitable cereal grains. Cultivation techniques of Pleurotus spp., Calocybe indica and Agaricus bisporus (long, short and indoor composting methods) with spawn and substrate preparation are discussed. Improved methodologies of mushroom culture preservation for a short or long period with advantages and limitations are discussed. Anticancer, anti-heart attack, anti-HIV, hypoglycaemic, hypotensive, hepato- and nephroprotective, antioxidant, immunomodulatory, cardiovascular, respiratory, anti-hepatotoxic, pharmacological properties (antifungal, antibacterial and antiviral), and production of novel bioactive molecules by mushrooms are highlighted.

Species Diversity of Rhizobia

The nitrogen-fixing bacteria that are capable of nodulating legumes are called rhizobia. Recycling of nitrogen through natural processes, i.e. symbiotic nitrogen fixation is considered as the most appropriated method for the long-term management of nutrients. These bacteria are heterogeneous assemblage of Gram-negative, aerobic, non-sporulating rod shaped, symbiont of legumes, and soil bacteria. The taxonomy and diversity of rhizobia were changed enormously in last two decades. Only about 20% of the total of about 20,000 species and 57% of about 750 genera of legume plants has been studied for nodulation. The species listed herein are authentically published names of rhizobia consisting 176 species spread over 15 genera and have been validated using molecular markers. There are many legumes and ecosystems that have not been analysed yet and then the number of rhizobial species and symbiovars are expected to increase in further. MultiLocus Sequence Analysis (MLSA) is currently being used for further segregation of species with accurate taxon. Consequently, whole genomes could be considered as the best alternative to studying diversity assessment and cataloguing of rhizobia species.

Role of Root Nodule Bacteria in Improving Soil Fertility and Growth Attributes of Leguminous Plants Under Arid and Semiarid Environments

The nitrogen fixation is a key factor in low-input agricultural systems to sustain long-term soil fertility. Plant–bacteria interactions in the rhizosphere are the determinants of plant health and soil fertility. The climatic conditions of arid and semiarid regions are often characterized by hot, dry summers, subhumid monsoon, and cold dry winter. The climatic conditions in this region restrict the buildup of soil organic matter and soils are generally deficient in nitrogen. Knowledge of rhizobial diversity from arid and semiarid areas is essential to improve their nutrient-poor fertility status. Soils of these regions often suffer from moisture stress, salinity, unfavorable pH, nutrient deficiency, mineral toxicity, temperature extremes, plant diseases, trace element deficiencies, etc., which inhibit nodulation and impose limitations on the vigor of the host legume. The tolerance to high levels of salinity and the survival and persistence in harsh desert conditions make these rhizobia highly valuable inocula to improve productivity of the leguminous plants cultivated under extreme environments. This functional diversity and tolerance to extreme environments displayed by efficient rhizobial isolates alone or co-inoculation with PGPR can be effectively utilized for improving legume crop production and productivity in arid and semiarid regions.

Diversity, Nitrogen fixation, and Biotechnology of Rhizobia from Arid Zone Plants

The climate of arid zones is often characterized as hot and dry summers, subhumid monsoon, and cold dry winters. Rhizobia have potential to restore soil fertility and sand dune stabilization in arid regions due to higher tolerance to salt stress, elevated temperature, and drought. The rhizobia have diverse and heterogeneous group divided into alpha- and beta-proteobacteria; nevertheless, they are united by their ability to form nodules on leguminous and non-leguminous plants. Due to nitrogen-fixing ability and potential to replace nitrogen fertilizers, rhizobia are among the most intensively studied groups of microorganisms; simultaneously, testing of nodulation by different bacteria led to the establishment of cross-inoculation groups. With the advent of modern biotechnological tools and techniques such as rDNA sequencing, 16S diversity, DNA–rRNA hybridizations, and rRNA catalogues, more diversity of rhizobia could be exposed. The rhizobia isolated from leguminous plants of arid region largely belong to Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium genera on the basis of morphophysiological and molecular characterization. The specific stress-tolerant traits of these bacteria can be exploited to mitigate climate resilience in the context of global warming.