Improvement of rooting and growth in kiwifruit (Actinidia deliciosa) cuttings with organic biostimulants.

Seaweed extracts have shown profoundly positive effects on crop growth, quality and reproduction in diverse agricultural and horticultural crops. Seaweed extracts can be used to promote the rooting and growth of cuttings in perennial fruit species like kiwifruit (Actinidia deliciosa). In this study, the cuttings were treated with 1, 5, 10 and 50% solutions of G Sap (Gracilaria edulis), K Sap (Kappaphycus alvarezii), AN (Ascophyllum nodosum), EM (Ecklonia maxima), HA (Humic acid) and control (water) for 6 h as base dipping. Subsequently, the treatments of G Sap, K Sap, AN, EM, HA and control were repeated every 15 days for a period of six months as application of 50 ml solutions in the potted cuttings. All the treatments exhibited significant effects on the rooting percent in all the kiwifruit cultivars, namely 'Monty', 'Abott', 'Hayward', 'Allison' and 'Bruno' (P ≤ 0.01) as compared to the control. Shoot and root growth parameters including leaf number per cutting, number of roots per cutting, number of branches, plant height, shoot diameter, root length, root diameter and root weight were all positively increased with the application of seaweed extracts (P ≤ 0.05). Cuttings treated with seaweed extract exhibited significantly higher levels of pigments (chlorophyll a, chlorophyll b and total carotenoids), metabolites (total carbohydrates and soluble phenols) and less electrolyte leakage as compared to the control cuttings. Significant positive and negative correlations were observed between biochemical parameters combined with plant nutrient concentration. Principal component analysis (PCA) revealed that PC1 and PC2 (first two principal components) accounted for 75% of the entire variation. While, PC1 accounted for 63% of the total variation, PC2 accounted for 11% of the total variation. The leaves and the roots of kiwifruit cultivar 'Hayward' treated with G Sap at 10%, K Sap at 10%, AN at 10%, EM at 10%, HA at 10% exhibited higher expression of all four root promoting candidate genes (GH3-3, LBD16, LBD29 and LRP1) compared to the control. Therefore, it can be concluded that, seaweed extract and humic acid can be used as a suitable alternative to synthetic hormones for promoting the rooting and growth of kiwifruit cuttings.

A Perspective on Current Flavivirus Vaccine Development: A Brief Review.

The flavivirus genus contains several clinically important pathogens that account for tremendous global suffering. Primarily transmitted by mosquitos or ticks, these viruses can cause severe and potentially fatal diseases ranging from hemorrhagic fevers to encephalitis. The extensive global burden is predominantly caused by six flaviviruses: dengue, Zika, West Nile, yellow fever, Japanese encephalitis and tick-borne encephalitis. Several vaccines have been developed, and many more are currently being tested in clinical trials. However, flavivirus vaccine development is still confronted with many shortcomings and challenges. With the use of the existing literature, we have studied these hurdles as well as the signs of progress made in flavivirus vaccinology in the context of future development strategies. Moreover, all current licensed and phase-trial flavivirus vaccines have been gathered and discussed based on their vaccine type. Furthermore, potentially relevant vaccine types without any candidates in clinical testing are explored in this review as well. Over the past decades, several modern vaccine types have expanded the field of vaccinology, potentially providing alternative solutions for flavivirus vaccines. These vaccine types offer different development strategies as opposed to traditional vaccines. The included vaccine types were live-attenuated, inactivated, subunit, VLPs, viral vector-based, epitope-based, DNA and mRNA vaccines. Each vaccine type offers different advantages, some more suitable for flaviviruses than others. Additional studies are needed to overcome the barriers currently faced by flavivirus vaccine development, but many potential solutions are currently being explored.

In silico and in vitro evaluation of silibinin: a promising anti-Chikungunya agent.

Chikungunya virus (CHIKV) infection and subsequent high patient morbidity is a global threat. The present study aimed to identify the potent antiviral agent against Chikungunya virus, with minimum in vitro cytotoxicity. CHIKV nsP4 3D structure was determined using the I-TASSER server followed by its refinement and pocket determination. Furthermore, high-throughput molecular docking was employed to identify candidate CHIKV nsP4 inhibitors in a library containing 214 compounds. The top ranked compound was evaluated further with various assays, including cytotoxicity, antiviral activity, time of drug addition, viral entry attachment, and microneutralization assays. High-throughput computational screening indicated silibinin to have the best interaction with CHIKV nsP4 protein, immature and mature glycoproteins with highest negative free binding energy, - 5.24 to - 5.86 kcal/mol, and the lowest inhibitory constant, 50.47 to 143.2 µM. Further in vitro analysis demonstrated silibinin could exhibit statistically significant (p < 0.05) dose-dependent anti-CHIKV activity within 12.5-100-µM concentrations with CC50 as 50.90 µM. In total, 50 µM silibinin interfered with both CHIKV attachment (75%) and entry (82%) to Vero cells. Time of addition assay revealed silibinin interfered with late phase of the CHIKV replication cycle. Microneutralization assay revealed that silibinin could inhibit clearing of 50% Vero cell monolayer caused by CHIKV-induced CPE at a minimum dose of 25 µM. These data indicated silibinin to be a promising candidate drug against CHIKV infection.

Dengue Fever and Severe Dengue in Barbados, 2008-2016.

Analysis of the temporal, seasonal and demographic distribution of dengue virus (DENV) infections in Barbados was conducted using national surveillance data from a total of 3994 confirmed dengue cases. Diagnosis was confirmed either by DENV-specific real time reverse transcriptase polymerase chain reaction (rRT-PCR), or non-structural protein 1 (NS1) antigen or enzyme linked immunosorbent assay (ELISA) tests; a case fatality rate of 0.4% (10/3994) was observed. The prevalence rate of dengue fever (DF) varied from 27.5 to 453.9 cases per 100,000 population among febrile patients who sought medical attention annually. DF cases occurred throughout the year with low level of transmission observed during the dry season (December to June), then increased transmission during rainy season (July to November) peaking in October. Three major dengue epidemics occurred in Barbados during 2010, 2013 and possibly 2016 with an emerging three-year interval. DF prevalence rate among febrile patients who sought medical attention overall was highest among the 10-19 years old age group. The highest DF hospitalisation prevalence rate was observed in 2013. Multiple serotypes circulated during the study period and Dengue virus serotype 2 (DENV-2) was the most prevalent serotype during 2010, whilst DENV-1 was the most prevalent serotype in 2013. Two DENV-1 strains from the 2013 DENV epidemic were genetically more closely related to South East Asian strains, than Caribbean or South American strains, and represent the first ever sequencing of DENV strains in Barbados. However, the small sample size (n = 2) limits any meaningful conclusions. DF prevalence rates were not significantly different between females and males. Public health planning should consider DENV inter-epidemic periodicity, the current COVID-19 pandemic and similar clinical symptomology between DF and COVID-19. The implementation of routine sequencing of DENV strains to obtain critical data can aid in battling DENV epidemics in Barbados.

The ankyrin repeat gene family in Capsicum spp: Genome-wide survey, characterization and gene expression profile.

The ankyrin (ANK) repeat protein family is largely distributed across plants and has been found to participate in multiple processes such as plant growth and development, hormone response, response to biotic and abiotic stresses. It is considered as one of the major markers of capsaicin content in pepper fruits. In this study, we performed a genome-wide identification and expression analysis of genes encoding ANK proteins in three Capsicum species: Capsicum baccatum, Capsicum annuum and Capsicum chinense. We identified a total of 87, 85 and 96 ANK genes in C. baccatum, C. annuum and C. chinense genomes, respectively. Next, we performed a comprehensive bioinformatics analysis of the Capsicum ANK gene family including gene chromosomal localization, Cis-elements, conserved motif identification, intron/exon structural patterns and gene ontology classification as well as profile expression. Phylogenetic and domain organization analysis grouped the Capsicum ANK gene family into ten subfamilies distributed across all 12 pepper chromosomes at different densities. Analysis of the expression of ANK genes in leaf and pepper fruits suggested that the ANKs have specific expression patterns at various developmental stages in placenta tissue. Our results provide valuable information for further studies of the evolution, classification and putative functions of ANK genes in pepper.

Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids.

KEY MESSAGE: Transcriptome landscape reveals the molecular mechanisms involved in the improvement of fruit traits by the grafting of watermelon and bottle gourd. Grafting has been used as a sustainable alternative for watermelon breeding to control soil-borne pathogens and to increase tolerance to various abiotic stresses. However, some reports have shown that grafting can negatively affect the quality of fruits. Despite several field studies on the effects of grafting on fruit quality, the regulation of this process at the molecular level has not been revealed. The aim of this study was to elucidate various molecular mechanisms involved in different tissues of heterografted watermelon and bottle gourd plants. Grafting with bottle gourd rootstock increased the size and rind thickness of watermelon fruits, whereas that with watermelon rootstock produced bottle gourd fruits with higher total soluble solid content and thinner rinds. Correspondingly, genes related to ripening, softening, cell wall strengthening, stress response and disease resistance were differentially expressed in watermelon fruits. Moreover, genes associated mainly with sugar metabolism were differentially expressed in bottle gourd fruits. RNA-seq revealed more than 400 mobile transcripts across the heterografted sets. More than half of these were validated from PlaMoM, a database for plant mobile macromolecules. In addition, some of these mobile transcripts contained a transfer RNA-like structure. Other RNA motifs were also enriched in these transcripts, most with a biological role based on GO analysis. This transcriptome study provided a comprehensive understanding of various molecular mechanisms underlying grafted tissues in watermelon.

Biology, Predatory Potential, Life Table, and Field Evaluation of Propylea dissecta (Coleoptera: Coccinellidae), Against Lipaphis erysimi (Hemiptera: Aphididae) on Broccoli.

Propylea dissecta (Mulsant) (Coleoptera: Coccinellidae) is one of the most promising ladybird beetle against many sucking pests. Predation rates, developmental biology, life table, and field assessment of this ladybird were examined against mustard aphid, Lipaphis erysimi (Kalt.) (Hemiptera: Aphididae), on broccoli. Data on the life history were collected at 23 ± 1°C and 70 ± 1% RH and were evaluated using the two-sex, age-stage life table. Results showed that the two-sex, age-stage life table-based net reproductive rate (R0) was 11.264 ± 6.197 offspring. The adult females lived longer (33.8 ± 2.356 d) than the adult males (32.2 ± 0.841 d). The fourth instar consumed most of L. erysimi (113.97 ± 5.76) compared to the other larval stages of the predator. Male (1,821) and female (2,673) consumed more aphids than larvae. The net consumption rate was 741.78 ± 89.91 aphids. Other aphidophagous predators such as Coccinella septempunctata L., Micraspis discolor (F.), Coccinella transversalis (F.), and syrphid (Diptera: Syrphidae) were also noted in broccoli. Our research showed that inoculative release of 150 or 200 adults per 1,000 m2 for two times on broccoli achieved a significant decrease in aphids L. erysimi and Brevicoryne brassicae L. (Hemiptera: Aphididae) (>95%). The release rate of 150 adults per 1,000 m2 for two times may, therefore, be recommended to manage the aphid population on broccoli.

Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp.

ATP-binding cassette (ABC) transporter genes act as transporters for different molecules across biological membranes and are involved in a diverse range of biological processes. In this study, we performed a genome-wide identification and expression analysis of genes encoding ABC transporter proteins in three Capsicum species, i.e., Capsicum annuum, Capsicum baccatum and Capsicum chinense. Capsicum is a valuable horticultural crop worldwide as an important constituent of many foods while containing several medicinal compounds including capsaicin and dihydrocapsaicin. Our results identified the presence of a total of 200, 185 and 187 ABC transporter genes in C. annuum, C. baccatum and C. chinense genomes, respectively. Capsaicin and dihydrocapsaicin content were determined in green pepper fruits (16 dpa). Additionally, we conducted different bioinformatics analyses including ABC genes classification, gene chromosomal location, Cis elements, conserved motifs identification and gene ontology classification, as well as profile expression of selected genes. Based on phylogenetic analysis and domain organization, the Capsicum ABC gene family was grouped into eight subfamilies. Among them, members within the ABCG, ABCB and ABCC subfamilies were the most abundant, while ABCD and ABCE subfamilies were less abundant throughout all species. ABC members within the same subfamily showed similar motif composition. Furthermore, common cis-elements involved in the transcriptional regulation were also identified in the promoter regions of all Capsicum ABC genes. Gene expression data from RNAseq and reverse transcription-semi-quantitative PCR analysis revealed development-specific stage expression profiles in placenta tissues. It suggests that ABC transporters, specifically the ABCC and ABCG subfamilies, may be playing important roles in the transport of secondary metabolites such as capsaicin and dihydrocapsaicin to the placenta vacuoles, effecting on their content in pepper fruits. Our results provide a more comprehensive understanding of ABC transporter gene family in different Capsicum species while allowing the identification of important candidate genes related to capsaicin content for subsequent functional validation.

Chikungunya virus: genomic microevolution in Eastern India and its in-silico epitope prediction.

This is the first study reporting whole genome sequences of two CHIKV strains (KJ679577 and KJ679578) isolated from Eastern Indian patients sera during 2010-2011 outbreak, both of which were of ECSA genotype, but from different subgroups: Indian Ocean outbreak and ECSA subtypes. Furthermore, viral sequences were analyzed using different in-silico approaches to identify potential genetic variations that might have functional implications on various aspects of virus replication, viral protein functionality, immunogenicity and transmission. Epitope prediction analysis revealed 70.9% increase in number of MHC Class-II interacting epitopes of KJ679578 and 25-28% increase in Class-I interacting epitopes of KJ679577 and KJ679578 compared to that of EF027141 (CHIKV of Asian genotype circulating in India during 1973, after which CHIKV infection disappeared from India for three decades). CHIKV peptides DLAKLAFKRSSKYDLECAQIPVHMKSDA and KVVLCGDPKQCGFFNMMQMKYNYNHNI were predicted to interact with maximum number of HLA Class-I (68 and 76.5%, respectively) and Class-II (47 and 100%, respectively) alleles present within Indian population with allele frequency of > 0.1 and were also recognized as predicted B-cell epitopes with BCPred score between 0.766 and 0.961 and with antigenicity ranging from 0.52 to 1.69; thus these peptides might be used to induce T- and B-cell-mediated immunity against CHIKV. Thus, the present study might help to bridge the gap between virus microevolution and its implication in host immunity by taking into account viral genetic and conformational changes. Predicted epitopes might be used as promising targets for peptide-based vaccine development and rapid diagnostics against CHIKV infection.

Association of toll-like receptor polymorphisms with susceptibility to chikungunya virus infection.

Chikungunya virus (CHIKV) infection leads to activation of innate immune response by triggering Toll-like receptor (TLR) pathways resulting in elevated cytokines and type-I interferon levels. Genetic variations of these genes may influence human CHIKV-susceptibility and disease progression. Present study aimed to identify role of TLR polymorphisms in CHIKV-susceptibility and their association with cytokines and clinical symptoms. This is the first study illustrating certain genotypes of TLR-7 and TLR-8 SNPs viz. CT(p = 0.002)]; rs3853839[GC(p < 0.001), CC(p = 0.039)] and rs3764879[GC(p < 0.001)] were considerably associated with CHIKV susceptibility. Increased risk of CHIKV infection among male patients with CC-genotype (rs179010) (p = 0.028) and female patients with GT-genotype (rs5741880) (p = 0.019) was observed. Significant higher IFN-α (P = 0.002) levels among chikungunya TNF-α (P = 0.034) patients was reported. Chikungunya patients with rs179010-CC genotype showed significantly high IFN-α level(p = 0.003). Thus, these TLR variants might act as potential prognostic biomarkers for predicting CHIKV susceptibility among uninfected individuals.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid diagnosis of chilli veinal mottle virus.

Chilli veinal mottle virus (ChiVMV) causes significant economic loss to chilli cultivation in northeastern India, as well as in eastern Asia. In this study, we have developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and specific diagnosis of ChiVMV. Amplification could be visualized after adding SYBR Green I (1000×) dye within 60 min under isothermal conditions at 63 °C, with a set of four primers designed based on the large nuclear inclusion protein (NIb) domain of ChiVMV (isolate KC-ML1). The RT-LAMP method was 100 times more sensitive than one-step reverse transcription polymerase chain reaction (RT-PCR), with a detection limit of 0.0001 ng of total RNA per reaction.

Identification and characterization of a distinct banana bunchy top virus isolate of Pacific-Indian Oceans group from North-East India.

Banana bunch top virus (BBTV) is considered to be a serious threat to banana production. A new isolate of the virus (BBTV-Umiam) was identified and characterized from local banana mats growing in mid-hills of Meghalaya in North-East India. The complete nucleotide sequence analysis revealed the presence of six full-length ssDNA components (DNA R, DNA U3, DNA S, DNA M, DNA C and DNA N) sharing major common region (CR-M) and a stem-loop common region (CR-SL). BBTV-Umiam showed a unique deletion of 20 nucleotides in the intergenic region of DNA R, the absence of predicted open reading frame (ORF) in DNA U3 and probability for a small ORF in DNA U3 expecting functional evidence at transcriptional level. Phylogenetic analysis based on 88 complete nucleotide sequence of BBTV DNA R available in GenBank generated two broad clusters of Pacific-Indian Oceans (PIO) and South-East Asian (SEA) groups including BBTV-Umiam within PIO cluster. However, BBTV-Umiam was identified as the most distinct member of the PIO group with 100% bootstrap support. This was further supported by the phylogenetic grouping of each genomic component of BBTV-Umiam at the distant end of PIO group during clustering of 21 complete BBTV sequences. BBTV-Umiam shared relatively less nucleotide identity with PIO group for each genomic component (85.0-95.4%) and corresponding ORF (93.8-97.5%) than that of earlier PIO isolates (91.5-99.6% and 96.0-99.3%, respectively). Recombination analysis revealed two intra-component and five inter-component recombination events in BBTV-Umiam, but none of them was unique. Moreover, the isolate was identified as major parental sequence for intra-component recombination event spanning the replication-associated protein encoding region in Tongan BBTV DNA R. The current study indicated differential evolution of BBTV in North-East India (Meghalaya). The natural occurrence of hybrids of Musa balbisiana and M. acuminata in this geographically isolated region could be the contributing factor in accumulating genetic distinctiveness in BBTV-Umiam which need further characterization.

Copy number variation of Chikungunya ECSA virus with disease symptoms among Indian patients.

After a gap of three decades, from 2005 onwards, a series of Chikungunya virus (CHIKV) outbreaks occurred worldwide. This study was performed to detect CHIKV infection, its genotype among symptomatic Eastern Indian patients and to analyze any association between the presence of CHIKV genome in patient body with appearance of disease symptoms (n = 199). Plasma-extracted viral RNA was reverse transcribed to cDNA and PCR-amplified followed by agarose gel electrophoresis. Viral load among CHIKV-positive patients was determined by real time RT-PCR. CHIKV-IgM in sera was detected by ELISA. Sequencing and phylogenetic analysis of plasma-extracted PCR products was done. CHIKV genome and IgM were detected among 65.3% (n = 130) and 41.2% (n = 82) patients respectively. Joint swelling was significantly associated with CHIKV infection (P-value: 0.0003). CHIKV PCR positive patients were grouped in two categories: Group-I: viral load <10(4) copies/ml and Group-II: viral load ≥10(4) copies/ml. Higher number of acute stage patients clustered in Group-II. Fever and joint swelling were significantly more prevalent among Group-II patients, whereas rash and diarrhoea among Group-I patients (P-value <0.05). Patient-isolated CHIKV sequences clustered with CHIKV ECSA genotypes in the phylogenetic tree, with two types of CHIKV strains found to circulate among them-as indicated by their different nucleotide sequences. This is the first study detecting the presence of CHIKV ECSA genotype among Eastern Indian patients. Fever and joint swelling might have appeared first followed by rash, diarrhea during disease progression-as indicated by CHIK viral load in patients. Thus, viral load can be used as unique diagnostic and prognostic marker of Chikungunya disease pathogenesis.

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India.

Pathogenic Klebsiella pneumoniae, resistant to beta-lactam and quinolone drugs, is widely recognized as important bacteria causing array of diseases. The resistance property is obtained by acquisition of plasmid encoded blaTEM, blaSHV, blaCTX-M, QNRA, QNRB and QNRS genes. The aim of this study was to document the prevalence and association of these resistant genes in K. pneumoniae infecting patients in India. Approximately 97 and 76.7 % of the 73 K. pneumoniae isolates showed resistance towards beta-lactam and quinolone drugs respectively. Bla genes were detected in 74 % of K. pneumoniae isolates; with prevalence in the following order: blaTEM > blaSHV > blaCTXM. QNR genes were detected in 67 % samples. Chi-square analysis revealed significant association between presence of bla and qnr genes in our study (P value = 0.000125). Sequence analysis of some blaTEM, blaSHV, blaCTX-M and QNRB PCR products revealed presence of blaTEM1 (GenBank accession: JN193522), blaTEM116 (JN193523 and JN193524), blaSHV11, blaCTXM72 variants (JF523199) and QNRB1 (JN193526 and JN193527) in our samples.