Host–microbiome interactions are specific and not random, making them defining entities for the host. The hypothesis proposed by various researchers earlier, that both plants and animals harbor specific inheritable core microbiome, is being augmented in the present study. Additionally, a case for using microbial fingerprint as a biomarker, not only for plant identification but also as a geographical indicator, has been investigated, taking Crocus sativus, saffron, as a study material. Crocus sativus, a monogenetic herb, on account of its male sterility and vegetative propagation, is reported to lack genome based molecular markers. Cormosphere microbiome (microbiome associated with corm) has been compared across three geographical locations, in two continents, to identify the core and unique microbiome, during the vegetative phase of its growth. Microbiome analysis done at phylum and genus level, using next generation sequencing technology, revealed that cormosphere at three locations harbored common phyla. At genus level, 24 genera were found common to all three geographical locations, indicating them to be part of the core microbiome of saffron. However, there were some bacterial genera unique to Kashmir, Kishtwar, and Morocco that can be used to develop microbial markers/geographical indicators for saffron grown in these regions. This is a preliminary study, indicating that the location specific bacterial community can be used to develop microbial barcodes but needs further augmentation with high coverage data from other saffron growing geographical regions.

Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the notable advances highlighted here include the development of transgene (DNA)-free genome plants, the availability of compatible nucleases, and the development of safe and effective CRISPR delivery vehicles for plant genome editing, multi-gene targeting and complex genome editing, base editing and prime editing to achieve more complex genetic engineering. Additionally, new avenues that facilitate fine-tuning plant gene regulation have also been addressed. In spite of the tremendous potential of CRISPR and other gene editing tools, major challenges remain. Some of the challenges are related to the practical advances required for the efficient delivery of CRISPR reagents and for precision genome editing, while others come from government policies and public acceptance. This review will therefore be helpful to gain insights into technological advances, its applications, and future challenges for crop improvement.

Native Bacillus sp. strain D5 coded as (Bar D5) has been isolated from the saffron corm that showed plant growth promotion (PGP) properties and also inhibits the growth of corm rot causing Fusarium oxysporum R1 (Fox R1) in-vitro. Bar D5 was more efficient PGP bacterium in comparison to earlier reported native bio-formulations by our group. Pot assays and field evaluation of Bar D5 confirmed its in-vivo efficacy for PGP traits and biocontrol activity as well. Pot trials were followed by field trials at traditional (Kishtwar) and non-traditional (R.S Pura) saffron cultivation areas in Jammu and Kashmir. At both places, Bar D5 bio-formulation treatment led to the increase in root number & length, shoot number & length, flower number and number & weight of daughter corms. Additionally, it also decreased the corm rot disease incidence significantly. Priming of corms with bio-formulation resulted in the reduction of pathogenic fungal load by three fold at the depth of corm sowing from ground level. The shelf life/viability of Bar D5 based bio-formulation was found to be 52% (viable spores) for one year at room temperature. Draft genome sequence of Bar D5 revealed the presence of genes necessary for PGP and biocontrol activity. Further, confirmation of gene sequences and annotation was done by amplification, re-sequencing and mapping of PGP and biocontrol genes on draft genome. Bar D5 based bio-formulation can be provided to companies/researchers interested in saffron cultivation or bio-formulation production for commercial exploitation, since saffron is grown as revenue crop across continents. The present study bridges the gap between genomics and its field application.

The corm rot of saffron caused by Fusarium oxysporum (Fox) has been reported to be the most destructive fungal disease of the herb globally. The pathogen, Fusarium oxysporum R1 (Fox R1) isolated by our group from Kashmir, India, was found to be different from Fusarium oxysporum f.sp. gladioli commonly reported corm rot agent of saffron. In the present study, Fox R1 was further characterized using housekeeping genes and pathogenicity tests, as Fusarium oxysporum R1 f.sp. iridacearum race 4. Though Fox R1 invaded the saffron plant through both corm and roots, the corm was found to be the preferred site of infection. In addition, the route of pathogen movement wastracked by monitoring visual symptoms, semi-quantitative PCR, quantitative-PCR (q-PCR), real-time imaging of egfp-tagged Fusarium oxysporum R1, and Fox R1 load quantification. This study is the first study of its kind on the bidirectional pathogenesis from corm to roots and vice-versa, as the literature only reports unidirectional upward movement from roots to other parts of the plant. In addition, the colonization pattern of Fox R1 in saffron corms and roots was studied. The present study involved a systematic elucidation of the mode and mechanism of pathogenesis in the saffron Fusarium oxysporum strain R1 pathosystem.

Earlier plant growth promoting rhizo-bacteria (PGPRs) were isolated from the plants, by cultivation based techniques and the interaction was mostly thought to be bilateral. The routine bilateral study, with no information on the associated microbiome, could be one of the reasons for the limited success of PGPRs in the field conditions. Keeping in view the role of PGPRs in rhizo-bacteriome on the growth and production of plant, the present study was aimed at studying the diversity of the rhizo-bacteriome of saffron grown across three geographical locations namely Kashmir, Kishtwar and Bengaluru. Variation in the rhizo-bacteriome of saffron growing across 10 different sites from 3 geographical locations was studied using 16S rDNA amplicon metagenomic sequencing. 16 bacterial phyla, 261 genera and 73 bacterial species were cataloged from all the rhizosphere samples. Proteobacteria was a dominant phylum in all the rhizosphere samples. Rhizo-bacteriome of saffron grown in Kishtwar was found to be significantly different from the rhizo-bacteriome of saffron grown in Kashmir and Bengaluru. Interestingly, the rhizo-bacteriome of saffron grown in Bengaluru was very similar to the saffron grown in Kashmir, thereby indicating that the rhizo-bacteriome in saffron is “plant driven” as the corm sown in Bengaluru were from Kashmir. Despite variation in rhizo-bacteriome, core rhizo-bacteriome in saffron was identified that was represented by 53 genera and eight bacterial species belonging to 11 phyla irrespective of their geographical distribution. In addition, 21 PGPRs were reported for the first time from the saffron rhizosphere. The high yielding saffron field Wuyan was found to have the highest number of PGPRs; this indicates that the presence of PGPR is important for yield enhancement than diversity. The two PGPR Rhizobium leguminosarum and Luteibacter rhizovicinus were reported from all the locations except Kishtwar that had escaped isolation in our previous attempts using cultivation based techniques. It is being proposed instead of going for random isolation and screening for PGPRs from plant rhizosphere, an alternate strategy using metagenomic cataloging of the rhizo-bacteriome community and cultivation of the dominant PGPR should be undertaken. This strategy will help in the selection of dominant PGPRs, specific to the plant in question.

Fusarium oxysporum causes corm rot in saffron (Crocus sativus L.), that is one of the most important fungal diseases impacting saffron yield globally. Despite the fact that the corm rot agent and its symptoms are widely known, little is known about the molecular basis of defense mechanism of saffron in response to Fusarium oxysporum infection. Therefore, the current study was initiated in order to identify differentially expressed genes in response to pathogen infection in saffron. The active participation of Mitogen Activated Kinase pathway (MAPK), Transcription factors (TFs), plant-hormone signalling, plant-pathogen interaction pathway and synthesis of PR proteins in defence of saffron against Fox R1 infection was revealed by Gene Ontology, KEGG pathway and MapMan analysis. In this study, the PR proteins had shown a robust antifungal activity. These findings revealed that the saffron has a powerful defense mechanism in the early stages of infection. In addition, fifty seven Fusarium oxysporum R1 genes linked to pathogenicity and virulence that expressed during the infection phase were also identified. Surprisingly, SIXgenes (secreted in the xylem) were not found in the current investigation, although these genes have been thoroughly described in other Fusarium oxysporum strains and are known to be one of the key virulence factors. Because saffron is a male sterile plant that can only be improved genetically by genome editing, this work will serve as a foundation for identifying genes that can be used to create saffron varieties resistant to Fox infection.

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Fusarium oxysporum has been reported to be the most devastating pathogen of Crocus sativus L., a commercially significant crop that yields the saffron spice. However, most of the pathogen isolations have been done from the diseased tissue, mostly from rotten corms, but no study has been conducted on diseased saffron fields. To fill the knowledge gap, the current study was carried out with the intention of recording the diversity of cultivable fungus species from saffron fields and screening them for pathogenicity towards saffron. The three study locations in Jammu and Kashmir, Srinagar (Pampore), Kishtwar, and Ramban, yielded a total of 45 fungal isolates. The internal transcribed spacer (ITS) of rDNA was used for the molecular identification. ITS rDNA-based sequence analysis classified all the operational taxonomic units (OTUs) into two phyla—Ascomycota (88.88%) and Mucoromycota (11.11%). Moreover, Fusarium (57.77%), Geotrichum (17.77%), Mucor (11.11%), Aspergillus (4.44%), Trichoderma (4.44%), Galactomyces (2.22%), and Colletotrichum (2.22%) all had different total abundances at the genus level. It was discovered that the saffron fields in Srinagar have fewer varied fungal species than the other two selected sites. All of the fungal isolates isolated including Fusarium solani, Aspergillus flavus, Trichoderma harzianum, Fusarium neocosmosporiellum, and Mucor circinelloides were pathogenic according to the pathogenicity test; however, injury to the saffron plant was found to be a must. These fungi were pathogenic in addition to F. oxysporum, which is well documented as a major cause of saffron corm rot diseases in Srinagar, but in the present study, injury was a must for F. oxysporum as well. The percentage disease severity index for both saffron roots and corms varied for each fungal isolate.