Effects of biocontrol Bacillus sp. strain D5 on the pathogenic Fusarium oxysporum R1 at the microscopic and molecular level in Crocus sativus L. (saffron) corm.

Corm rot of saffron caused by Fusarium oxysporum is a major threat to saffron cultivation the world over. To minimize the ill effects of chemical fungicides, attention has been shifted to the use of biocontrol agents for disease management in a sustainable way. In saffron, various biocontrol agents against corm rot disease have been reported and characterized but no study has been done so far to understand their interaction at the molecular level. The present study was conducted to unravel the mechanism of action of an already characterized native biocontrol agent i.e. Bacillus sp. strain D5 (Bar D5) against F. oxsporum R1 (Fox R1) in the saffron corm. The growth inhibition of Fox R1 was observed in vitro and in planta (saffron corm) by real time imaging. Bacillus sp. strain D5 reduced Fox R1 load in infected corms by 50% as quantified by q-PCR and the colony-forming unit method. Comparative transcriptome analysis revealed upregulation and downregulation of various Fox R1 genes in presence of Bar D5. The genes related to carbon metabolism, cell wall and membrane synthesis, and growth of Fox R1 were significantly downregulated in Bar D5-primed and Fox R1-inoculated corms as compared to only Fox R1-inoculated corms.

Molecular warfare between pathogenic Fusarium oxysporum R1 and host Crocus sativus L. unraveled by dual transcriptomics.

KEY MESSAGE: Phenylpropanoid biosynthesis and plant-pathogen interaction pathways in saffron and cell wall degrading enzymes in Fusarium oxysporum R1 are key players involved in the interaction. Fusarium oxysporum causes corm rot in saffron (Crocus sativus L.), which is one of the most devastating fungal diseases impacting saffron yield globally. Though the corm rot agent and its symptoms are known widely, little is known about the defense mechanism of saffron in response to Fusarium oxysporum infection at molecular level. Therefore, the current study reports saffron-Fusarium oxysporum R1 (Fox R1) interaction at the molecular level using dual a transcriptomics approach. The results indicated the activation of various defense related pathways such as the mitogen activated protein kinase pathway (MAPK), plant-hormone signaling pathways, plant-pathogen interaction pathway, phenylpropanoid biosynthesis pathway and PR protein synthesis in the host during the interaction. The activation of pathways is involved in the hypersensitive response, production of various secondary metabolites, strengthening of the host cell wall, systemic acquired resistance etc. Concurrently, in the pathogen, 60 genes reported to be linked to pathogenicity and virulence has been identified during the invasion. The expression of genes encoding plant cell wall degrading enzymes, various transcription factors and effector proteins indicated the strong pathogenicity of Fusarium oxysporum R1. Based on the results obtained, the putative molecular mechanism of the saffron-Fox R1 interaction was identified. As saffron is a male sterile plant, and can only be improved by genetic manipulation, this work will serve as a foundation for identifying genes that can be used to create saffron varieties, resistant to Fusarium oxysporum infection.

Usage of postpartum intrauterine contraceptive device (postpartum-IUD) after an increase in the institutional delivery rate in India: time to re-visit the effect of timing of counselling on its acceptance.

PURPOSE: While increase in institutional deliveries brings an opportunity to counsel women for postpartum family planning (PPFP), its uptake remains low. Reasons for poor acceptance of postpartum intrauterine contraceptive device (postpartum-IUD), and its relation with the timing of counselling need to be investigated. METHODS: Women attending the antenatal clinic, reporting in labour, and within 48 h of delivery respectively were invited to participate. Eligible women were asked about awareness and choice for PPFP. After counselling, acceptance for PPFP was compared with the baseline. Acceptance and continuation of postpartum-IUD were compared between women counselled in the antenatal, intrapartum, and postpartum periods. RESULTS: Only 23% of 360 women were aware of postpartum-IUD. After counselling, acceptance for PPFP increased from 14% to 97% and for postpartum-IUD, from 0.5% to 33.9%. Acceptance of postpartum-IUD among women counselled in the antenatal, intrapartum and postpartum period was 45%, 35% and 21.7% respectively. Acceptance was higher among the antenatal-counselling group than the postpartum-counselling group (OR 0.45; CI 0.22-0.94; p = 0.03). CONCLUSION: Counselling, irrespective of its timing, improves acceptance for PPFP. Acceptance and continuation of postpartum-IUD are higher following counselling in antenatal period. All eligible women should be counselled irrespective of 'when' they approach the facility.

Acceptance for postpartum-IUD is maximum when women are counselled in the antenatal period. With a surge in institutional deliveries, the opportunity to counsel women in the intrapartum and postpartum period should not be missed as this also increases acceptance for PPFP and postpartum-IUD.

Injury and not the pathogen is the primary cause of corm rot in Crocus sativus (saffron).

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.

Invasion and Colonization of Pathogenic Fusarium oxysporum R1 in Crocus sativus L. during Corm Rot Disease Progression.

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.

CRISPR-Based Genome Editing Tools: Insights into Technological Breakthroughs and Future Challenges.

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.

Field evaluation of PGP Bacillus sp. strain D5 native to Crocus sativus, in traditional and non traditional areas, and mining of PGP genes from its genome.

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.