Identification of a novel mitovirus in grapevine through high-throughput sequencing.

BACKGROUND: Transcriptome data from a plant sample frequently include numerous reads originating from RNA virus genomes that were concurrently isolated during RNA preparation. These high-throughput sequencing reads from the virus can be assembled to form a new sequence for the plant RNA genome. METHODS AND RESULTS: Here, we identify putative novel mitovirus, grapevine mitovirus 1 (GMV1) through high-throughput sequencing (HTS) of grapevine rootstocks (Vitis spp.), and the identified virus was confirmed using virus-specific primers in RT-PCR assay. The genomic RNA of GMV1 encodes complete open reading frame (ORF) of 2,496 nucleotides (nts) in length. RNA-dependent RNA polymerase (RdRp) encoded by the viral genome contained one RdRp conserved domain. BLASTx analysis of GMV1 genome showed sequence identity of 33.18-56.75% with the existing mitovirus sequences. Phylogenetic analysis based on genome sequences showed that GMV1 clustered in a distinct clade to other mitoviruses. CONCLUSION: Grapevine mitovirus 1 represents a newly discovered species within the Unuamitovirus genus of the Mitoviridae family, targeting fungal mitochondria. While the majority of recognized mitoviruses typically lack a functional RdRp as per the plant mitochondrial genetic code, GMV1 encodes a complete RdRp in accordance with both fungal and plant mitochondrial genetic codes.

Development of Simplified Recombinase Polymerase Amplification Assay for Rapid and Robust Detection of Citrus Yellow Vein Clearing Virus.

Citrus is an economically important fruit crop, belongs to family Rutaceae, cultivated commercially in over 130 countries, which holds a leading profitable position in the international market. The most important citrus varieties are mandarins, oranges, lemons, sweet limes, grapefruits and pomelos. Citrus yellow vein clearing virus (CYVCV) is an important graft transmissible plant pathogen known to reduce productivity of citrus fruits due to its predominant association and widespread occurrence. Requirement of fast, reliable, efficient & economical CYVCV indexing assay is a prerequisite for production of healthy planting material. Currently, nucleic acid isolation and thermal cycler-based assay available for CYVCV indexing is a cumbersome lab intensive method. The present study was undertaken to develop and validate reverse transcription-recombinase polymerase amplification (RT-RPA) assay requiring no tedious RNA isolation, separate cDNA synthesis and costlier instrument like thermo-cycler. Optimized RT-RPA assay was able to amplify CYVCV up to 10-7 dilution (equivalent to 0.1 pg/µl) with the prepared templates of both RNA and crude saps and showed higher sensitivity in detection of CYVCV infection in field samples as compared to the conventional RT-PCR. Developed RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus tristeza virus, citrus exocortis viroid and huanglongbing). RT-RPA using crude leaf sap as template is quite simple, robust, highly sensitive, time and cost effective; therefore, it can be used in resource constrained laboratories as screening tool, for field surveys and on-site testing programs in farms, nurseries and biosecurity. Present study, first time reports the development, optimization and validation of crude sap-based RT-RPA assay for the detection of CYVCV infection in citrus plants namely; Kinnow mandarin, Mosambi and Grape fruit.

Simple template-based reverse transcription-recombinase polymerase amplification assay for routine diagnosis of citrus tristeza virus.

This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32 min), although the best conditions were 38°C for 30 min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.

Growth kinetics, improved plant growth and alleviation of water stress in tomato by water stress tolerant bacteria.

Plants are subject to a variety of abiotic stresses contributed to yield losses of up to 50%, posing a significant challenge to global food production. To cope with drought stress, of 205 bacterial cultures investigated for moisture stress tolerant potential, 16 cultures showed promising results in improving the majority of plant growth ameliorating activities under water stress and non-stress conditions. Growth kinetics and plant growth ameliorating activities declined significantly with the increase in water stress level. Most of the isolates tolerant to water stress were Streptomyces and Pseudomonas species. Of these, four strains with the best results were selected for growing tomato under water stress conditions. The imposition of water stress severely inhibited the growth of tomato plants. However, bacterial strains alleviated the stress and enhanced plant growth performance. Antioxidant activity showed a promising result of protection from reactive oxygen species produced in plants because of water stress. Plants treated with bioinoculants also exhibited a substantial decline in lipid peroxidation. Water stress significantly reduced the yield of tomato. However, bioinoculants treated plants demonstrated significantly higher yields than untreated plants. Nutrient uptake and fruit quality also improved in the treated plants. Experiments point to the scope of developing a microbial formulation to alleviate water stress in higher plants.

Anticarcinogenic and Antioxidant Action of an Edible Aquatic Flora Jussiaea repens L. Using In Vitro Bioassays and In Vivo Zebrafish Model.

Oxidative stress is the major cause of many health conditions, and regular consumption of antioxidants helped to encounter and prevent such oxidative stress-related diseases. Due to safety concerns over long-term uses of synthetic antioxidants, natural antioxidants are more preferred. The purpose of this study is to investigate the antioxidant and anticancer activities of Jussiaea repens L., a wild edible flora found in Manipur, India. The antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Ferric reducing antioxidant power (FRAP) assay and DNA-nicking assay. The anticancer activity was tested using five cancer lines viz., SKOV3 cells (ovarian), HeLa (cervical), MDA-MB-231 (breast), PANC-1 (pancreatic), and PC3 (prostate). The toxicity, developmental effect, antiproliferative activity was further tested using zebrafish embryos. The methanolic plant extract had higher polyphenol content than flavonoids. The in vitro study demonstrated a promising antioxidant capacity and DNA protection ability of this plant. The extract also showed cytotoxic activity against SKOV3, HeLa, MDA-MB-23, and PANC-1 cancer cell lines. The in vivo studies on zebrafish embryos demonstrated the extract's ability to suppress the developmental process and elicited more cytotoxicity to cancer cells than developing zebrafish embryos. Moreover, the in vivo studies on zebrafish embryos also indicated the antiproliferative activity of J. repens L. extract.

Valorization of chicken feather waste into bioactive keratin hydrolysate by a newly purified keratinase from Bacillus sp. RCM-SSR-102.

The objective of this study is the characterization of a keratinase from Bacillus sp.RCM-SSR-102 and its application in the preparation of keratin hydrolysate from chicken feather waste. The purified KER102 keratinase was characterized as a serine-metallo protease having a molecular weight of 30 kDa with optimum pH and temperature of 10 and 50 °C respectively. The keratinase could retain 98% activity at pH 10 and above and 55% activity at 20% salt concentration. The KER102 keratinase was found to be stable in the presence of oxidizing agents, surfactants and organic solvents. The keratinase could also hydrolyze both soluble and insoluble complex protein substrates. The KER102 keratinase could hydrolyze up to 5% (w/v) feather releasing 1.7 ± 0.19 mg/mL soluble peptides. The feather keratin hydrolysate (FKH) had both antioxidant and antityrosinase activity. The IC50 value of FKH in 2, 2-diphenyl 1-picrylhydrazyl (DPPH) radical scavenging activity (1.02 ± 0.01 mg/mL), 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity (20 ± +00.04 µg/mL) and anti-tyrosinase activity (1.2 ± 0.22 mg/mL) was recorded. The FKH also had DNA protecting ability against oxidative damage. Antioxidant and anti-tyrosinase compounds have potential applications in the pharmaceutical and cosmeceutical industry. Hence, the purified keratinase can be a potential candidate for the production of antioxidant and antityrosinase compounds from chicken feather waste.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

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.

Subpopulation level variation of banana streak viruses in India and common evolution of banana and sugarcane badnaviruses.

Genome sequences of three episomal Banana streak MY virus (BSMYV) isolates sampled from triploid banana hybrids (Chini Champa: AAB; Malbhog: AAB and Monthan: ABB), grown in North-East and South India are reported in this study by sequence-independent improved rolling circle amplification (RCA). RCA coupled with restriction fragment length polymorphism revealed diverse restriction profiles of five BSMYV isolates. Nucleotide substitution rates of BSMYV subpopulation and Banana streak OL virus subpopulation was 7.13 × 10(-3) to 1.59 × 10(-2) and 2.65 × 10(-3) to 5.49 × 10(-3), respectively, for the different coding regions. Analysis of the genetic diversity of banana and sugarcane badnaviruses revealed a total of 32 unique recombination events among banana and sugarcane badnaviruses (inter BSV-SCBV), in addition to the extensive recombination with in banana streak viruses and sugarcane bacilliform viruses (intra-BSV and intra-SCBV). Many unique fragments were shown to contain similar ruminant sequence fragments which indicated the possibility that the two groups of badnaviruses or their ancestors to colonise same host before making the host shift. The distribution of recombination events, hot-spots (intergenic region and C-terminal of ORF3) as well as cold-spots (distributed in ORF3) displayed the mirroring of recombination traces in both group of badnaviruses. These results support the hypothesis of relatedness of banana and sugarcane badnaviruses and the host and geographical shifts that followed the fixation of the species complex appear to be a recent event.

A novel method for detecting conformation-dependent ß-glucans-single, double, and triple-helical from Shiitake mushroom.

This pioneering study explores the structural intricacies of therapeutic ß-glucan in Shiitake (Lentinula edodes), i.e. Lentinan (LNT). Lentinan, a neutral polysaccharide [ß-(1,3; 1,6) glucan], exists in three forms; single, double, and triple-helical, but conformation-dependent bioactivity studies are lacking. In this context, we meticulously assessed indigenous Shiitake accessions from Northeast India, unveiling the conformational spectrum of LNT through an innovative pipeline. The experiment approached the simultaneous estimation of total glucan (TG), triple helical glucan (THG), and single-double helical glucan (SDG). Profiling revealed the exceptional LNT content in DMRO-623 (TG: 46.74%, SDG: 9.34%, THG: 37.39%) which emerged as the highest documented to date. Beyond the culinary delight, this research and the novel approach to LNT quantification will create a pivotal platform for advanced mushroom research, offering prospects for novel discoveries, innovative applications, and therapeutic potential.

Mixed infection of two mandariviruses identified by high-throughput sequencing in Kinnow mandarin and development of their specific detection using duplex RT-PCR.

In the current study, high-throughput sequencing (HTS) was used to identify viruses associated with the Kinnow mandarin (Citrus reticulata) plants exhibiting yellow vein clearing, mottling, and chlorosis symptoms at experimental farm of ICAR-Indian Agricultural Research Institute, New Delhi, India. During November 2022, leaf samples of symptomatic and asymptomatic Kinnow mandarin trees were collected, subjected to HTS and one of the representative symptomatic samples was subjected to leaf-dip electron microscopy (EM). In the EM results, flexuous virus particles typical of mandarivirus were observed. Ribosomal RNA was depleted from total RNA of pooled samples and RNA sequencing was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs, which were annotated through BLASTn using database of plant viruses/viroids reference genomes (NCBI). Results of assembled contigs revealed near-complete genomes of two mandariviruses, i.e., citrus yellow vein clearing virus (CYVCV) and citrus yellow mottle-associated virus (CiYMaV). The values of fragments per kilo base transcript length per million fragments mapped estimation indicated the dominance of CYVCV in HTS data and it was also confirmed through krona plot distribution of viruses in the pooled samples. A rapid and reliable duplex RT-PCR assay was also developed and standardized for the simultaneous detection of both CYVCV and CiYMaV in a pooled Kinnow mandarin sample. The developed duplex RT-PCR was then validated for the presence of these viruses in individual Kinnow mandarin samples. The specificity and sensitivity results confirmed that primers were highly specific to their targets and able to detect viruses up to 10-2 dilutions of RNA in standard and duplex RT-PCR. Therefore, the developed rapid duplex RT-PCR can be used for virus indexing and production of virus-free Kinnow mandarin plants for certification programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04011-9.

Development and application of crude sap-based recombinase polymerase amplification assay for the detection and occurrence of grapevine geminivirus A in Indian grapevine cultivars.

Geminiviruses are known to infect several fields and horticultural crops around the globe. Grapevine geminivirus A (GGVA) was reported in the United States in 2017, and since then, it has been reported in several countries. The complete genome recovered through high-throughput sequencing (HTS)-based virome analysis in Indian grapevine cultivars had all of the six open reading frames (ORFs) and a conserved nonanucleotide sequence 5'-TAATATTAC-3' similar to all other geminiviruses. Recombinase polymerase amplification (RPA), an isothermal amplification technique, was developed for the detection of GGVA in grapevine samples employing crude sap lysed in 0.5 M NaOH solution and compared with purified DNA/cDNA as a template. One of the key advantages of this assay is that it does not require any purification or isolation of the viral DNA and can be performed in a wide range of temperatures (18°C-46°C) and periods (10-40 min), which makes it a rapid and cost-effective method for the detection of GGVA in grapevine. The developed assay has a sensitivity up to 0.1 fg µl-1 using crude plant sap as a template and detected GGVA in several grapevine cultivars of a major grapevine-growing area. Because of its simplicity and rapidity, it can be replicated for other DNA viruses infecting grapevine and will be a very useful technique for certification and surveillance in different grapevine-growing regions of the country.

DNA protection ability, antihyperglycemic potential and in-vivo toxicity assessment of arrowleaf tearthumb (Persicaria sagittata L.), a neglected aquatic plant species of Eastern Asia.

Persicaria sagittata L. (common name arrowleaf tearthumb, American) is an herbaceous edible plant with characteristics sessile leaves mainly found in wetland areas of North America and Eastern Asia. In Eastern Himalayan Region of India, the ethnic communities consumed this plant as vegetables. The present investigation suggests the plant is endowed with bioactive compounds having potential DNA protection ability and antihyperglycemic activity. The DNA nicking assay revealed that the methanolic extract of this plant has the potential to protect plasmid DNA against hydroxyl damage. The α-glucosidase and α-amylase inhibitory assay of this methanolic extract suggest more effectiveness in inhibition of α-amylase than the α-glucosidase. Further, proximate composition, micronutrient, total phenolic and flavonoid content of this underutilised aquatic plant was determined. And lastly the in-vivo cytotoxicity study of Persicaria sagittata L. plant extract suggest that the plant is less toxic to in-vivo system.

Reactive oxygen species turnover, phenolics metabolism, and some key gene expressions modulate postharvest physiological deterioration in cassava tubers.

Rapid postharvest physiological deterioration (PPD) in cassava (Manihot esculenta Crantz) tuber is a significant concern during storage. The freshly harvested tubers start spoiling within 24 to 72 h. Accumulation of H2O2 is one of the earliest biochemical events that occurred during PPD, which was detected using the 3,3 diaminobenzidine (DAB) in two contrast cassava genotypes, MNP Local A (29-57 µg g-1) and Sree Prakash (64-141 µg g-1). Accumulating the fluorescence hydroxycoumarin compounds emitted by the cassava tubers observed under an ultraviolet (UV) lamp showed significant variations at 0, 3, 6, 9, 12, and 15 days of storage. The total phenolics and carotenoids significantly and negatively correlated with PPD progression; however, the anthocyanin and flavonoids positively correlated with the PPD-anchored ROS accumulation. The primary compound, Phthalic acid, di(2-propylpentyl) ester, was identified in both the cassava tubers, Sree Prakash (57.21 and 35.21%), and MNP Local A (75.58 and 60.21%) at 0, and 72 h of PPD, respectively. The expression of PPD-associated genes APX-2, APX-3, PAL, and AP was higher at 6-12 days of PPD, which signified the synthesis of ROS turnover and phenylpropanoid biosynthesis. A significant, strong, and positive correlation was established between the secondary metabolites and PPD signaling gene expression, which was inversely correlated with hydroxycoumarin and H2O2 accumulation. MNP Local A tubers exhibited longer storage life of 15 days with a low PPD score, higher metabolites synthesis, and gene expression. The PPD-resistant lines may be used to augment cassava breeding strategies for large-scale commercial and industrial use.

Bioactivity and nutritional quality of nutgall (Rhus semialata Murray), an underutilized fruit of Manipur.

Introduction: Underutilized fruits plays a significant role in socio economic, cultural, nutritional and ethnomedicinal status of tribal people. However, scientific studies on the nutritional and other pharmaceuticals/biological activities of these fruits are meagre. Hence, the present study dealt with the quantification of nutritional quality and deciphering the bioactivity of nutgall (Rhus semialata Murray syn. Rhus chinensis Mill.), an underutilized fruit crop mainly found in foothill tracks of Eastern Himalaya, India, China, Japan, Korea and other South East Asian countries. Methods: The Rhus semialata Murray fruits were collected from five different locations in Purul sub-division, Senapati district, Manipur, India. The nutritional composition of the fruit pulp was analysed. Further the fruit pulp was extracted in methanol and water. The methanol and water extracts were studied for bioactivity properties such as antioxidant, antihyperglycemic, antihypertensive, antihyperuricemia, anti-tyrosinase, and antimicrobial activity. Results and discussion: The fruit was rich in essential fatty acids. The presence of linoleic and oleic acids, along with traces of docosahexaenoic acid and eicosapantaenoic acid, revealed the potential food value of the fruit. 59.18% of the total amino acid composition of the protein present was constituted by essential amino acids. The IC50 value of methanolic extract (MExt) and Water extract (WExt) of the fruit were recorded as 4.05 ± 0.22 and 4.45 ± 0.16 µg/mL, respectively, in the DPPH assay and 5.43 ± 0.37 and 11.36 ± 2.9 µg/mL, respectively, in the ABTS assay as compared to Ascorbic acid (3 and 5.4 µg/mL in DPPH and ABTS assay, respectively). The CUPRAC assay also showed a high antioxidant potential of MExt and WExt (1143.84 ± 88.34 and 456.53 ± 30.02 mg Ascorbic Acid Equivalent/g, respectively). MExt and WExt of the fruit were more active against α-glucosidase (IC50 of 1.61 ± 0.34 and 7.74 ± 0.54 µg/ mL, respectively) than α-amylase enzyme (IC50 14.15 ± 0.57 and 123.33 ± 14.7 µg/mL, respectively). In addition, the methanolic fruit extract showed low to moderate pharmacological potential in terms of antihypertensive (Angiotensin converting enzyme-I inhibition), antihyperuricemia (xanthine oxidase inhibition), anti-tyrosinase, and antimicrobial activity. The IC50 values of angiotensin-converting enzyme I inhibition, xanthine oxidase inhibition and tyrosinase inhibition were recorded as 13.35 ± 1.21 mg/mL, 93.16 ± 4.65 mg/mL, and 862.7 ± 12.62 µg/mL, respectively. The study evidently indicates that nutgall fruit is a potential source of phytonutrients, bestowed with commercially exploitable, multifaceted health benefits.

A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis.

Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150-450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management.

DNA Barcoding and ITS2 Secondary Structure Predictions in Taro (Colocasia esculenta L. Schott) from the North Eastern Hill Region of India.

Taro (Colocasia esculenta L. Schott, Araceae), an ancient root and tuber crop, is highly polygenic, polyphyletic, and polygeographic in nature, which leads to its rapid genetic erosion. To prevent the perceived loss of taro diversity, species discrimination and genetic conservation of promising taro genotypes need special attention. Reports on genetic discrimination of taro at its center of origin are still untapped. We performed DNA barcoding of twenty promising genotypes of taro indigenous to the northeastern hill region of India, deploying two chloroplast-plastid genes, matK and rbcL, and the ribosomal nuclear gene ITS2. The secondary structure of ITS2 was determined and molecular phylogeny was performed to assess genetic discrimination among the taro genotypes. The matK and rbcL genes were highly efficient (>90%) in amplification and sequencing. However, the ITS2 barcode region achieved significant discrimination among the tested taro genotypes. All the taro genotypes displayed most similar sequences at the conserved matK and rbcL loci. However, distinct sequence lengths were observed in the ITS2 barcode region, revealing accurate discriminations among the genotypes. Multiple barcode markers are unrelated to one another and change independently, providing different estimations of heritable traits and genetic lineages; thus, they are advantageous over a single locus in genetic discrimination studies. A dynamic programming algorithm that used base-pairing interactions within a single nucleic acid polymer or between two polymers transformed the secondary structures into the symbol code data to predict seven different minimum free energy secondary structures. Our analysis strengthens the potential of the ITS2 gene as a potent DNA barcode candidate in the prediction of a valuable secondary structure that would help in genetic discrimination between the genotypes while augmenting future breeding strategies in taro.

In silico molecular modelling, structural dynamics simulation and characterization of antifungal nature of ß-glucosidase enzyme from Sechium edule.

ß-glucosidase is an enzyme that has ability to cleave ß-glycosidic bonds present in oligosaccharides and glycoconjugates. They are known to be present across all domains of living organism and have important roles in many biological processes including plant defense mechanism. In the present study, a ß-glucosidase enzyme identified from seeds of Sechium edule was characterized using various bioinformatics tools. A homology model (SeBG) was generated using a ß-glucosidase crystal structure from Oryza sativa (PDB ID: 3PTK) as template. In silico structural binding studies on putative ß-glucosidase protein revealed a stable and strong interaction indicative of higher GOLD fitness score with the substrates: p-nitrophenyl-ß-d-glucopyranoside (pNPG), laminarin, chitotriose, N-acetylglucosamine and N-acetylmuramic acid suggesting its possible role in broad spectrum antifungal and antimicrobial activity. Assessment of the in vitro enzyme activity with pNPG showed a Km and Vmax values of 2.7 mM and 22 µMmin-1mL-1mg-1, respectively. While, the in vitro enzyme activity with laminarin showed a Km and Vmax values of 0.31 mM and 0.043 µMmin-1mL-1mg-1. The broad spectrum activity of the protein shown in our result indicates SeBG as a promising biocontrol agent against phytopathogens.Communicated by Ramaswamy H. Sarma.

Candidatus Liberibacter asiaticus manipulates the expression of vitellogenin, cytoskeleton, and endocytotic pathway-related genes to become circulative in its vector, Diaphorina citri (Hemiptera: Psyllidae).

Citrus greening disease or huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) limits citrus production worldwide. CLas is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) in a persistent-propagative manner. Understanding the molecular interaction between CLas and ACP and interrupting the interrelationship can provide an alternative to insecticides for managing citrus greening disease. Transcriptome analysis of ACP in response to CLas showed differential expression of 3911 genes (2196 upregulated, and 1715 downregulated) including the key genes of ACP involved in cytoskeleton synthesis and nutrition-related proteins, such as vitellogenins, extensin, laminin, tropomyosin, troponin C, and flightin. Majority of the differentially expressed genes were categorized under molecular functions followed by cellular components and biological processes. KEGG pathway analysis showed differential regulation of carbohydrate, nucleotide, and energy metabolic pathways, the endocytotic pathway, and the defense-related pathways. Differential regulation of genes associated with the key pathways might favour CLas to become systemic and propagate in its insect vector. The study provides an understanding of genes involved in circulation of CLas in ACP. The candidate genes involved in key physiological processes and CLas transmission by ACP would be potential targets for sustainable management of ACP and CLas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02641-x.

CRISPR-Cas-Led Revolution in Diagnosis and Management of Emerging Plant Viruses: New Avenues Toward Food and Nutritional Security.

Plant viruses pose a serious threat to agricultural production systems worldwide. The world's population is expected to reach the 10-billion mark by 2057. Under the scenario of declining cultivable land and challenges posed by rapidly emerging and re-emerging plant pathogens, conventional strategies could not accomplish the target of keeping pace with increasing global food demand. Gene-editing techniques have recently come up as promising options to enable precise changes in genomes with greater efficiency to achieve the target of higher crop productivity. Of genome engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins have gained much popularity, owing to their simplicity, reproducibility, and applicability in a wide range of species. Also, the application of different Cas proteins, such as Cas12a, Cas13a, and Cas9 nucleases, has enabled the development of more robust strategies for the engineering of antiviral mechanisms in many plant species. Recent studies have revealed the use of various CRISPR-Cas systems to either directly target a viral gene or modify a host genome to develop viral resistance in plants. This review provides a comprehensive record of the use of the CRISPR-Cas system in the development of antiviral resistance in plants and discusses its applications in the overall enhancement of productivity and nutritional landscape of cultivated plant species. Furthermore, the utility of this technique for the detection of various plant viruses could enable affordable and precise in-field or on-site detection. The futuristic potential of CRISPR-Cas technologies and possible challenges with their use and application are highlighted. Finally, the future of CRISPR-Cas in sustainable management of viral diseases, and its practical utility and regulatory guidelines in different parts of the globe are discussed systematically.