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.

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.

Metatranscriptomic insight into the possible role of clay microbiome in skin disease management.

Even though the scientific documentation is limited, microbiome of healing clay is gradually gaining attention of the scientific community, as a therapeutic force playing an indispensable role in skin disease management. The present study explores the metatranscriptome profile of the Chamliyal clay, widely known for its efficacy in managing various skin problems, using Illumina NextSeq sequencing technology. The gene expression profile of the clay microbial community was analyzed through SEED subsystems of the MG-RAST server. Due to the unavailability of metatranscriptomic data on other therapeutic clays, Chamliyal's profile was compared to non-therapeutic soils, as well as healthy and diseased human skin microbiomes. The study identified Firmicutes, Proteobacteria, and Actinobacteria as the primary active microbial phyla in Chamliyal clay. These resemble those abundant in a healthy human skin microbiome. This is significant as lower levels of these phyla in the skin are linked to inflammatory skin conditions like psoriasis. Interestingly, pathogenic microbes actively metabolizing in the clay were absent. Importantly, 6% of the transcripts annotated to sulfur and iron metabolism, which are known to play a major role in skin disease management. This study provides the most comprehensive and a novel overview of the metatranscriptome of any of the healing clay available worldwide. The findings offer valuable insights into the clay microbiome's potential in managing skin disorders, inspiring future endeavors to harness these insights for medical applications.

Exploring the Potential of 87Sr/86Sr Isotope Ratio with Strontium and Rubidium Levels to Assess the Geographic Origin of Saffron.

Saffron is regarded as the most expensive spice, mainly because of its laborious harvest. Only a few countries dominate the global saffron market, with Iran producing by far the most saffron, and the saffron production of all other countries thus being much smaller. However, the respective national production (not only of saffron) is usually preferred by local consumers with respect to foreign products and often has a higher price. Cases of saffron with mislabeled geographic origin have repeatedly occurred. Thus, to protect local saffron production, control of the declared geographic origin is required. In the present case, differentiation of the geographic origin by 87Sr/86Sr is performed. The results show the saffron of several countries of origin to vary within the range of marine carbonates; however, saffron samples of Moroccan and Indian origin mainly show elevated 87Sr/86Sr values. Within the Indian saffron samples, one sample from Kishtwar Valley can be differentiated from the Kashmir saffron samples. The results are thus promising, especially when using the combination of Sr and Rb concentrations to differentiate geographic origin whenever the regions are of homogenous bedrock geology within and of different geology between the regions. However, the reported findings need to be checked and confirmed by further and additional saffron samples.

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.

Insights from the analysis of draft genome sequence of Crocus sativus L.

Saffron (Crocus sativus L.) is the low yielding plant of medicinal and economic importance. Therefore, it is of interest to report the draft genome sequence of C. sativus. The draft genome of C. sativus has been assembled using Illumina sequencing and is 3.01 Gb long covering 84.24% of genome. C. sativus genome annotation identified 53,546 functional genes (including 5726 transcription factors), 862,275 repeats and 964,231 SSR markers. The genes involved in the apocarotenoids biosynthesis pathway (crocin, crocetin, picrocrocin, and safranal) were found in the draft genome analysis.

Comprehensive account of present techniques for in-field plant disease diagnosis.

The early detection of plant pathogens is an appropriate preventive strategy for the management of crop yield and quality. For this reason, effective diagnostic techniques and tools, which are simple, specific, rapid and economic, are needed to be developed. Although several such technologies have been developed still most of them suffer one or the other limitation. Major limitations of the widely used diagnostic methods are requirement of trained staff and laboratory setup. Development of point-of-care diagnostic devices (handy portable devices) that require no specialized staff and can directly be used in fields is need of the hour. The aim of this review is to compile the information on current promising techniques that are in use for plant-pathogen diagnosis. Additionally, it focuses on the latest in-field pathogen diagnostic techniques with associated advantages and limitations.

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.

Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L.

Efficient transformation system for genetic improvement is essential in Crocus sativus, as it lacks sexual reproduction. This is the first report wherein an efficient protocol is developed for the transformation of Crocus sativus L. by Agrobacterium rhizogenes strain ARqua1 with a transformation efficiency of 78.51%. The ARqua1 strain harboring both Ri plasmid and binary vector plasmid pSITE-4NB, and marker genes for red fluorescent protein (RFP) and a ß-glucuronidase (GUS) reporter gene were used for selection. Transformation was confirmed by RFP signal, GUS reporter assay and polymerase chain reaction (PCR) analysis of the test samples after 21 days post inoculation. These results confirm the establishment of protocol for hairy root transformation in C. sativus that can be further used for gene transfer or gene editing in Crocus for its genetic improvement.

Metagenomic insights into the fungal assemblages of the northwest Himalayan cold desert.

Psychrophilic fungi are a critical biotic component in cold deserts that serves a central role in nutrient recycling and biogeochemical cycles. Despite their ecological significance, culture-independent studies on psychrophilic mycobiome are limited. In the present study, the fungal diversity patterns across the Drass, an Indian cold desert in the Himalaya, were indexed by targeted amplicon pyrosequencing (ITS). In the Drass dataset, Ascomycota was represented by 92 genera, while 22 genera represented Basidiomycota. The most abundant genus was Conocybe (20.46%). Most of the identified genera were reported in the literature to be prolific extracellular hydrolytic enzyme producers. To identify whether the Drass fungal assemblages share similarities to other cold deserts, these were further compared to Antarctic and Arctic cold deserts. Comparative analysis across the three cold deserts indicated the dominance of Dikarya (Ascomycota and Basidiomycota). The observed alpha diversity, Shannon index as well as Pielou's evenness was highest in the Antarctic followed by Drass and Arctic datasets. The genera Malassezia, Preussia, Pseudogymnoascus, Cadophora, Geopora, Monodictys, Tetracladium, Titaea, Mortierella, and Cladosporium were common to all the cold deserts. Furthermore, Conocybe was represented predominantly in Drass. Interestingly, the genus Conocybe has not been previously reported from any other studies on Antarctic or Arctic biomes. To the best of our knowledge, this is the first fungal metagenome study in Drass soil. Our analysis shows that despite the similarities of low temperature among the cold deserts, a significant differential abundance of fungal communities prevails in the global cold deserts.

Phylogenetic diversity and metabolic potential of microbiome of natural healing clay from Chamliyal (J&K).

Clay therapy for skin disease treatment is an ancient practice popular worldwide as a cheap alternative to pharmaceutical products. Effectiveness of clay against skin problems has been linked to its mineral composition and to microbial activity. The clay-water paste of a holy shrine Chamliyal in the Jammu region of J&K, India is used as an ointment to treat different skin disorders particularly psoriasis. Using the 16 SrDNA amplicon pyrosequencing and whole-metagenome direct shotgun Illumina sequencing, microbial phylogeny and potential metabolic functions were catalogued for Chamliyal's clay. Microbial diversity profile of the Chamliyal's clay is similar to other medicinal clays, particularly Dead Sea; there is some uniqueness as well. Although Proteobacteria, Actinomycetes and Firmicutes are common inhabitants of all the clay types, sulphur- and iron-reducing bacteria like Deferribacterales are particular to clays used for skin healing. In the present study it is proposed that healing properties of clay may be due to the microbes and microbial genes associated with metabolism of minerals like iron and sulphur, that lead to mineral acquisition in the Chamliyal's clay.

Cloning and characterization of thermo-alkalistable and surfactant stable endoglucanase from Puga hot spring metagenome of Ladakh (J&K).

A thermo-alkalistable and surfactant stable endoglucanase (PHS) gene consisting of 554 amino acids was identified from metagenomic library of Puga hot spring using functional screening. PHS gene was overexpressed and purified to homogeneity using affinity chromatography The purified PHS protein presented a single band of 60kDa on the SDS-PAGE gel and zymogram. The recombinant PHS exhibited activity over a broad range of pH and temperature with optima at pH 8.0 and 65°C, respectively and having optimum stability at 60°C and pH 8.0, respectively. The recombinant PHS showed highest substrate specificity using CMC (218.4U/mg) as compared with Barley ß-glucan (89.2U/mg) and Avicel (0.8U/mg). The Km and Vmax of recombinant PHS for CMC were 3.85mg/ml and 370.37µmolmin-1mg-1, respectively. The activity of the recombinant PHS was enhanced by treatment with 10mM non-ionic detergents such as Tween 20, Tween 40, Tween 80, Triton X- 100 and PEG and was inhibited by CTAB, SDS. Its functionality was stable in the presence of Fe3+ but inhibited by Cu2+, Hg2+, Mn2+ and Zn2+. These properties make PHS endoglucanase a potential candidate for use in laundry, textile,paper and pulp industries.

High Throughput Sequencing: An Overview of Sequencing Chemistry.

In the present century sequencing is to the DNA science, what gel electrophoresis was to it in the last century. From 1977 to 2016 three generation of the sequencing technologies of various types have been developed. Second and third generation sequencing technologies referred commonly to as next generation sequencing technology, has evolved significantly with increase in sequencing speed, decrease in sequencing cost, since its inception in 2004. GS FLX by 454 Life Sciences/Roche diagnostics, Genome Analyzer, HiSeq, MiSeq and NextSeq by Illumina, Inc., SOLiD by ABI, Ion Torrent by Life Technologies are various type of the sequencing platforms available for second generation sequencing. The platforms available for the third generation sequencing are Helicos™ Genetic Analysis System by SeqLL, LLC, SMRT Sequencing by Pacific Biosciences, Nanopore sequencing by Oxford Nanopore's, Complete Genomics by Beijing Genomics Institute and GnuBIO by BioRad, to name few. The present article is an overview of the principle and the sequencing chemistry of these high throughput sequencing technologies along with brief comparison of various types of sequencing platforms available.

Comparative Metagenomics Reveal Phylum Level Temporal and Spatial Changes in Mycobiome of Belowground Parts of Crocus sativus.

Plant-fungal associations have been explored by routine cultivation based approaches and cultivation based approaches cannot catalogue more than 5% of fungal diversity associated with any niche. In the present study, an attempt has been made to catalogue fungal diversity associated with belowground parts i.e. rhizosphere and cormosphere, of Crocus sativus (an economically important herb) during two growth stages, using cultivation independent ITS gene targeted approach, taking bulk soil as reference. The 454 pyrosequencing sequence data analysis suggests that the fungal diversity was niche and growth stage specific. Fungi diversity, in the present case, was not only different between the two organs (roots and corm) but the dominance pattern varies between the cormosphere during two growth stages. Zygomycota was dominant fungal phylum in the rhizosphere whereas Basidiomycota was dominant in cormosphere during flowering stage. However in cormosphere though Basidiomycota was dominant phylum during flowering stage but Zygomycota was dominant during dormant stage. Interestingly, in cormosphere, the phyla which was dominant at dormant stage was rare at flowering stage and vice-versa (Basidiomycota: Flowering = 93.2% Dormant = 0.05% and Zygomycota: Flowering = 0.8% Dormant = 99.7%). At genus level, Rhizopus was dominant in dormant stage but was rare in flowering stage (Rhizopus: Dormant = 99.7% Flowering = 0.55%). This dynamics is not followed by the bulk soil fungi which was dominated by Ascomycota during both stages under study. The genus Fusarium, whose species F. oxysporum causes corm rot in C. sativus, was present during both stages with slightly higher abundance in roots. Interestingly, the abundance of Rhizopus varied a great deal in two stages in cormosphere but the abundance of Fusarium was comparable in two growth stages (Bulk soil Flowering = 0.05%, Rhizosphere Flowering = 1.4%, Cormosphere Flowering = 0.06%, Bulk soil Dormant = 2.47% and cormosphere dormant = 0.05%). This is the first report on the fungal diversity associated with the root of Crocus sativus and first report on the fungi associated with corm of any plant with the temporal and spatial variation in the fungal community structure.

Cellulolytic Activity of Thermophilic Bacilli Isolated from Tattapani Hot Spring Sediment in North West Himalayas.

Eight thermophilic bacterial strains were isolated from Tattapani Hot spring and screened for various hydrolytic enzymes including cellulases. The isolated bacterial strains were identified as Geobacillus thermodenitrificans IP_WH1(KP842609), Bacillus licheniformis IP_WH2(KP842610), B. aerius IP_WH3(KP842611), B. licheniformis IP_WH4(KP842612), B. licheniformis IP_60Y(KP842613), G. thermodenitrificans IP_60A1(KP842614), Geobacillus sp. IP_60A2(KP842615) and Geobacillus sp. IP_80TP(KP842616) after 16S ribotying. Out of the eight isolates Geobacillus sp. IP_80TP grew best at 80 °C whereas rest of the isolates showed optimal growth at 60 °C. G. thermodenitrificans IP_WH1 produced a thermotolerant cellulase with maximum activity at 60 °C.

Bacterial diversity of Drass, cold desert in Western Himalaya, and its comparison with Antarctic and Arctic.

Drass is the coldest inhabited place in India and the second coldest, inhabited place in the world, after Siberia. Using the 16SrDNA amplicon pyrosequencing, bacterial diversity patterns were cataloged across the Drass cold desert. In order to identify the ecotype abundance across cold desert environment, bacterial diversity patterns of Drass were further compared with the bacterial diversity of two other cold deserts, i.e., Antarctic and Arctic. Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Gemmatimonadetes were among the highly abundant taxonomic groups present across all the three cold deserts and were designated as the core phyla. However, Firmicutes, Nitrospirae, Armatimonadetes (former candidate division OP10), Planctomycetes, TM7, Chloroflexi, Deinococcus-Thermus, Tenericutes and candidate phyla WS3 were identified as rare phyla in Drass, Antarctic and Arctic samples. Differential abundance patterns were also computed across all the three samples, i.e., Acidobacteria (32.1 %) were dominant in Drass and Firmicutes (52.9 ± 17.6 %) and Proteobacteria (42 ± 1.3 %) were dominant in Antarctic and Arctic reference samples, respectively. Alpha diversity values Shannon's (H) and Simpson's (1-D) diversity indices were highest for Antarctic samples, whereas richness estimators (ACE and Chao1) were maximum for Drass soil suggesting greater species richness in bacterial communities in Drass than the Antarctic and Arctic samples.

Isolation and in silico characterization of novel esterase gene with ß-lactamase fold isolated from metagenome of north western Himalayas.

An esterase-producing clone Aph2 was isolated from the Apharwat soil metagenomic library, a mountain peak in NW Himalayas. ORF 2 (Est Ac) of clone Aph2 corresponds to 271 aa protein and showed 26 % sequence similarity to carboxylesterase gene of Synechococcus sp. JA-2-3B. Est Ac contains nucleophilic Ser in S68-X-X-K71 motif of ß-lactamases with Tyr Y103. The conserved sequences are common with family VIII carboxylesterase and class C ß-lactamase sequences. Phylogenetic analysis revealed that Est Ac sequence is closely related to esterase than to ß-lactamases. In silico 3D protein structure of Est Ac was generated using MODELLER software (9.10 version). Model was generated on the basis of carboxylesterase template (PDB:1CI8) of Est B (Burkholderia gladioli) and the stereochemical parameters of the model generated were satisfactory. Docking with diisopropyl-fluorophosphate confirmed catalytic activity of Ser68 present in S-X-X-K motif.

Draft Genome Sequence of Plant Growth-Promoting Bacillus amyloliquefaciens Strain W2 Associated with Crocus sativus (Saffron).

Bacillus sp. strain W2 is a plant growth-promoting rhizobacterium isolated from saffron fields of Kashmir, India. Here, we report the draft genome sequence (3.9 Mb) of Bacillus amyloliquefaciens strain W2 having 65 contigs (3, 997, 511 bp), 4,163 coding sequences, and an average 46.45% GC content. Despite the 99% identity of the 16S rRNA gene with that of Bacillus amyloliquefaciens subsp. plantarum FZB42, the genome comparison revealed that only 48.7% of the W2 genome has homology with that of FZB42.

Draft Genome Sequence of Pseudomonas sp. Strain JMM, a Sediment-Hosted Environmental Isolate.

Pseudomonas sp. strain JMM was isolated from the sediments of a natural water reservoir (pH, 6 to 7) located at Chambyal village in Samba district of Jammu and Kashmir, India. Here we report the annotated draft genome sequence of strain JMM having 52 contigs with 5,884 genes and an average G+C content of 66.5%.