Geographical Expansion of Avian Metapneumovirus Subtype B: First Detection and Molecular Characterization of Avian Metapneumovirus Subtype B in US Poultry.

Avian metapneumovirus (aMPV), classified within the Pneumoviridae family, wreaks havoc on poultry health. It typically causes upper respiratory tract and reproductive tract infections, mainly in turkeys, chickens, and ducks. Four subtypes of AMPV (A, B, C, D) and two unclassified subtypes have been identified, of which subtypes A and B are widely distributed across the world. In January 2024, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the US. Metagenomics sequencing of selected tissue and swab samples confirmed the presence of aMPV subtype B. Subsequently, all samples were screened using an aMPV subtype A and B multiplex real-time RT-PCR kit. Of the 221 farms, 124 (56%) were found to be positive for aMPV-B. All samples were negative for subtype A. Six whole genomes were assembled, five from turkeys and one from chickens; all six assembled genomes showed 99.29 to 99.98% nucleotide identity, indicating a clonal expansion event for aMPV-B within the country. In addition, all six sequences showed 97.74 to 98.58% nucleotide identity with previously reported subtype B sequences, e.g., VCO3/60616, Hungary/657/4, and BR/1890/E1/19. In comparison to these two reference strains, the study sequences showed unique 49-62 amino acid changes across the genome, with maximum changes in glycoprotein (G). One unique AA change from T (Threonine) to I (Isoleucine) at position 153 in G protein was reported only in the chicken aMPV sequence, which differentiated it from turkey sequences. The twelve unique AA changes along with change in polarity of the G protein may indicate that these unique changes played a role in the adaptation of this virus in the US poultry. This is the first documented report of aMPV subtype B in US poultry, highlighting the need for further investigations into its genotypic characterization, pathogenesis, and evolutionary dynamics.

Effect of silencing Thrips palmi Btk29A and COL3A1 on fitness and virus acquisition.

Thrips palmi (Thysanoptera: Thripidae) is a major agricultural pest infesting over 200 plant species. Along with direct injury caused by feeding, T. palmi spreads several orthotospoviruses. Groundnut bud necrosis orthotospovirus (GBNV, family Tospoviridae, genus Orthotospovirus) is the predominant orthotospovirus in Asia, vectored by T. palmi. It is responsible for almost 89 million USD losses in Asia annually. Several transcripts of T. palmi related to innate immune response, receptor binding, cell signaling, cellular trafficking, viral replication, and apoptosis are responsive to the infection of orthotospoviruses in thrips. Expression of T. palmi tyrosine kinase Btk29A isoform X1 (Btk29A) and collagen alpha-1(III) chain-like (COL3A1) are significantly regulated post-GBNV and capsicum chlorosis orthotospovirus infection. In the present study, T. palmi Btk29A and COL3A1 were silenced and the effect on virus titer and fitness was assessed. The expression of Btk29A and COL3A1 was significantly reduced by 3.62 and 3.15-fold, respectively, 24 h post-dsRNA exposure. Oral administration of Btk29A and COL3A1 dsRNAs induced 60 and 50.9% mortality in T. palmi. The GBNV concentration in T. palmi significantly dropped post-silencing Btk29A. In contrast, the silencing of COL3A1 led to an increase in GBNV concentration in T. palmi compared to the untreated control. To the best of our knowledge, this is the first report on the effect of silencing Btk29A and COL3A1 on the fitness and GBNV titer in T. palmi.

Effect of silencing Bemisia tabaci TLR3 and TOB1 on fitness and begomovirus transmission.

Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important invasive pests worldwide. It infests several vegetables, legumes, fiber, and ornamental crops. Besides causing direct damage by sucking plant sap, B. tabaci is the principal vector of begomoviruses. Chilli leaf curl virus (ChiLCV, Begomovirus) transmitted by B. tabaci is a major constraint in chilli production. B. tabaci genes associated with metabolism, signaling pathways, cellular processes, and organismal systems are highly enriched in response to ChiLCV infection. The previous transcriptome study suggested the association of B. tabaci Toll-like receptor 3 (TLR3) and transducer of erbB2.1 (TOB1) in ChiLCV infection. In the present study, B. tabaci TLR3 and TOB1 were silenced using double-stranded RNA (dsRNA) and the effect on fitness and begomovirus transmission has been reported. Oral delivery of dsRNA at 3 µg/mL reduced the expression of B. tabaci TLR3 and TOB1 by 6.77 and 3.01-fold, respectively. Silencing of TLR3 and TOB1 induced significant mortality in B. tabaci adults compared to untreated control. The ChiLCV copies in B. tabaci significantly reduced post-exposure to TLR3 and TOB1 dsRNAs. The ability of B. tabaci to transmit ChiLCV also declined post-silencing TLR3 and TOB1. This is the first-ever report of silencing B. tabaci TLR3 and TOB1 to induce mortality and impair virus transmission ability in B. tabaci. B. tabaci TLR3 and TOB1 would be novel genetic targets to manage B. tabaci and restrict the spread of begomovirus.

MicroRNA Identification, Target Prediction, and Validation for Crop Improvement.

Micro-RNAs (mi-RNAs) are regulatory elements that play a vital role in the growth, development, and metabolic regulation of plants. In current research, the isolation of miRNAs is a tedious and difficult task using in vitro methods. However, recent exploration into the remarkably highly conserved nature of nucleotide sequences of miRNAs assists in the identification of miRNAs in plant species through homologous approaches. Here, we describe the in silico-based method for identification of miRNAs from the EST database which is emerging as a faster and more reliable approach along with the development of miRNA-SSR markers. This approach has the potential to accelerate research into the regulation of gene expression in various plant species such as tea, potato, tomato, tobacco, and orphan crops like cluster bean.

Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications.

Nucleic acid extraction is the first and foremost step in molecular biology studies. Extraction of DNA from small, soft-bodied insects is often time-consuming and costly. A fast, easy, and cost-effective DNA extraction method with greater yield and purity of DNA would aid in the rapid diagnostics, screening of large populations, and other routine PCR-based applications. The present study evaluated and standardized a rapid and zero-cost DNA extraction from soft-bodied small insects for routine molecular studies. Five rapid DNA extraction methods viz. extraction in sterile distilled water (SDW), 1X phosphate-buffered saline (PBS, pH 7.4), 1.4 M sodium chloride (NaCl), 20 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), and elution from blotted nitrocellulose membrane (NCM) were compared with standard CTAB extraction buffer and DNeasy® Blood and Tissue Kit methods. The average yield, purity, storage stability, time, and cost of extraction were assessed for all the methods and compared. A method of DNA extraction by simply crushing the soft-bodied insects in SDW was ideal in terms of yield, purity, storability, and performing routine PCR-based applications including detection of pathogens from vector species. The extraction could be accomplished in 2.5 min only with zero-reagent cost. The method would be useful in rapid molecular diagnostics and screening large populations of soft-bodied insects.

In silico Identification of miRNAs and Their Targets in Cluster Bean for Their Role in Development and Physiological Responses.

Cluster bean popularly known as "guar" is a drought-tolerant, annual legume that has recently emerged as an economically important crop, owing to its high protein and gum content. The guar gum has wide range of applications in food, pharma, and mining industries. India is the leading exporter of various cluster bean-based products all across the globe. Non-coding RNAs (miRNAs) are involved in regulating the expression of the target genes leading to variations in the associated pathways or final protein concentrations. The understanding of miRNAs and their associated targets in cluster bean is yet to be used to its full potential. In the present study, cluster bean EST (Expressed Sequence Tags) database was exploited to identify the miRNA and their predicted targets associated with metabolic and biological processes especially response to diverse biotic and abiotic stimuli using in silico approach. Computational analysis based on cluster bean ESTs led to the identification of 57 miRNAs along with their targets. To the best of our knowledge, this is the first report on identification of miRNAs and their targets using ESTs in cluster bean. The miRNA related to gum metabolism was also identified. Most abundant miRNA families predicted in our study were miR156, miR172, and miR2606. The length of most of the mature miRNAs was found to be 21nt long and the range of minimal folding energy (MFE) was 5.8-177.3 (-kcal/mol) with an average value of 25.4 (-kcal/mol). The identification of cluster bean miRNAs and their targets is predicted to hasten the miRNA discovery, resulting in better knowledge of the role of miRNAs in cluster bean development, physiology, and stress responses.

Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi.

Thrips cause considerable economic losses to a wide range of food, feed, and forest crops. They also transmit several plant viruses. Being cryptic, it is often difficult to distinguish thrips species in crops and large consignments by conventional methods. Melon thrips (Thrips palmi Karny, Thysanoptera: Thripidae) is an invasive insect pest of vegetables, legumes, and ornamentals besides being vector to several viruses. It poses a threat to domestic and international plant biosecurity and can invade and establish in new areas. Here, we report a polymerase spiral reaction (PSR)-based isothermal assay for rapid, sensitive, specific, low-cost, and on-site detection of T. palmi. To the best of our knowledge, this is the first application of PSR in the identification of any insect species. A primer pair designed based on 3'-polymorphism of mtCOIII region can specifically identify T. palmi without any cross-reactivity with predominant thrips species. The assay uses crude lysate of a single thrips saving time and reagents involved in nucleic acid extraction. The presence of T. palmi is visualized by the appearance of bright fluorescence under ultraviolet light or a change in reaction color thus avoiding gel electrophoresis steps. The entire process can be completed in 70 min on-site using only an ordinary water bath. The assay is sensitive to detecting as little as 50 attograms of T. palmi template. The assay was validated with known thrips specimens and found to be efficient in diagnosing T. palmi under natural conditions. The described method will be useful for non-expert personnel to detect an early infestation, accidental introduction to a new area, restrict the spread of diseases and formulate appropriate management strategies.

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi.

Thrips palmi (Thysanoptera: Thripidae) is the predominant tospovirus vector in Asia-Pacific region. It transmits economically damaging groundnut bud necrosis virus (GBNV, family Tospoviridae) in a persistent propagative manner. Thrips serve as the alternate host, and virus reservoirs making tospovirus management very challenging. Insecticides and host plant resistance remain ineffective in managing thrips-tospoviruses. Recent genomic approaches have led to understanding the molecular interactions of thrips-tospoviruses and identifying novel genetic targets. However, most of the studies are limited to Frankliniella species and tomato spotted wilt virus (TSWV). Amidst the limited information available on T. palmi-tospovirus relationships, the present study is the first report of the transcriptome-wide response of T. palmi associated with GBNV infection. The differential expression analyses of the triplicate transcriptome of viruliferous vs. nonviruliferous adult T. palmi identified a total of 2,363 (1,383 upregulated and 980 downregulated) significant transcripts. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed the abundance of differentially expressed genes (DEGs) involved in innate immune response, endocytosis, cuticle development, and receptor binding and signaling that mediate the virus invasion and multiplication in the vector system. Also, the gene regulatory network (GRN) of most significant DEGs showed the genes like ABC transporter, cytochrome P450, endocuticle structural glycoprotein, gamma-aminobutyric acid (GABA) receptor, heat shock protein 70, larval and pupal cuticle proteins, nephrin, proline-rich protein, sperm-associated antigen, UHRF1-binding protein, serpin, tyrosine-protein kinase receptor, etc., were enriched with higher degrees of interactions. Further, the expression of the candidate genes in response to GBNV infection was validated in reverse transcriptase-quantitative real-time PCR (RT-qPCR). This study leads to an understanding of molecular interactions between T. palmi and GBNV and suggests potential genetic targets for generic pest control.

Promising versions of a commercial pearl millet hybrid for terminal drought tolerance identified through MAS.

Extreme climatic conditions like drought are a major threat to global food production. Terminal drought stress causes severe yield losses in pearl millet. Development of climate-resilient varieties/hybrids can minimize the yield losses to the farmers caused due to climatic extremes. In the present study, marker-assisted selection (MAS) was employed with an aim to develop improved version of HHB 226 by introgression of QTLs for terminal drought stress tolerance into the male parent of the hybrid (HBL 11). HBL 11 (recurrent parent) was crossed with PRLT 2 (donor) to develop F1 and backcrossed four times to raise BC4F1 and further selfed twice to raise BC4F3. Four polymorphic SSR markers were used to track the QTL introgressed lines in each subsequent generation until BC4F2. The recurrent parent genome recovery was assessed using 25 polymorphic SSRs. Morpho-physiological analysis of BC4F3 generation at field-level under terminal drought stress conditions showed that the QTL introgressed lines showed higher, grain yield, 1000-seed weight, relative water content (%), and lower electrolyte leakage (%) than the recurrent parent. Line number 63 performed best with all the four foreground markers, 97.20% recurrent parent genome recovery, 7.27 g 1000-seed weight, 73.27% relative water content, 65.06% electrolyte leakage, 0.58 (fv/fm) chlorophyll fluorescence, and 53.25 g grain yield per plant. Finally, the Improved version of HHB 226 was developed by using the Improved HBL 11 developed through MAS. Besides this, HBL 11 is the male parent of other commercial hybrids like HHB 223 and HHB 197 as well making Improved HBL 11 an asset to improve these pearl millet hybrids.

Frontiers Approaches to the Diagnosis of Thrips (Thysanoptera): How Effective Are the Molecular and Electronic Detection Platforms?

Thrips are insect pests of economically important agricultural, horticultural, and forest crops. They cause damage by sucking plant sap and by transmitting several tospoviruses, ilarviruses, carmoviruses, sobemoviruses, and machlomoviruses. Accurate and timely identification is the key to successful management of thrips species. However, their small size, cryptic nature, presence of color and reproductive morphs, and intraspecies genetic variability make the identification of thrips species challenging. The use of molecular and electronic detection platforms has made thrips identification rapid, precise, sensitive, high throughput, and independent of developmental stages. Multi-locus phylogeny based on mitochondrial, nuclear, and other markers has resolved ambiguities in morphologically indistinguishable thrips species. Microsatellite, RFLP, RAPD, AFLP, and CAPS markers have helped to explain population structure, gene flow, and intraspecies heterogeneity. Recent techniques such as LAMP and RPA have been employed for sensitive and on-site identification of thrips. Artificial neural networks and high throughput diagnostics facilitate automated identification. This review also discusses the potential of pyrosequencing, microarrays, high throughput sequencing, and electronic sensors in delimiting thrips species.

High-Throughput Phenotyping: A Platform to Accelerate Crop Improvement.

Development of high-throughput phenotyping technologies has progressed considerably in the last 10 years. These technologies provide precise measurements of desired traits among thousands of field-grown plants under diversified environments; this is a critical step towards selection of better performing lines as to yield, disease resistance, and stress tolerance to accelerate crop improvement programs. High-throughput phenotyping techniques and platforms help unraveling the genetic basis of complex traits associated with plant growth and development and targeted traits. This review focuses on the advancements in technologies involved in high-throughput, field-based, aerial, and unmanned platforms. Development of user-friendly data management tools and softwares to better understand phenotyping will increase the use of field-based high-throughput techniques, which have potential to revolutionize breeding strategies and meet the future needs of stakeholders.

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses.

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3' polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management.

A multiplex PCR assay for rapid identification of major tospovirus vectors reported in India.

BACKGROUND: To date, four thrips vectors have been reported to transmit five different tospoviruses in India. Their identification at an early stage is crucial in formulating appropriate pest management strategies. Since morphometric key-based thrips identification based on the adult stage is time-consuming, there is a need to develop diagnostic tools which are rapid, accurate, and independent of developmental stages. Here, we report a multiplex PCR assay to identify four major thrips vectors viz. Thrips palmi, T. tabaci, Scirtothrips dorsalis, and Frankliniella schultzei present in India. RESULTS: Cytochrome oxidase subunit III and internal transcribed spacer region 2 were utilized to design species-specific primers. Of 38 pairs of primers tested, primer pairs AG35F-AG36R, AG47F-AG48R, AG87F-AG88R, and AG79F-AG80R amplified 568 bp, 713 bp, 388 bp, and 200 bp products from the DNA templates of T. palmi, S. dorsalis, T. tabaci, and F. schultzei, respectively at same PCR conditions. The specificity of the primer pairs was validated with a large number of known specimens and no cross-reactivity was observed with other thrips species. The multiplex PCR assay with a cocktail of all the four primer pairs detected four thrips vectors efficiently and could discriminate all of them concurrently in a single reaction. CONCLUSION: The multiplex PCR reported in this study could identify the major thrips vectors reported in India. The assay will be useful in ascertaining distribution profile of major thrips vectors, disease epidemiology, screening large samples, and quarantine.

Exposure to watermelon bud necrosis virus and groundnut bud necrosis virus alters the life history traits of their vector, Thrips palmi (Thysanoptera: Thripidae).

Thrips palmi transmits the tospoviruses watermelon bud necrosis (WBNV) and groundnut bud necrosis virus (GBNV) in persistent propagative way. Little is known about the T. palmi-WBNV and -GBNV relationship. In this study, we report the effects of WBNV and GBNV infection on the life history traits of T. palmi. Both WBNV and GBNV had some negative effects on the adult life span, fecundity and survival of T. palmi as compared to non-exposed T. palmi. Tospovirus exposure favoured a female-biased ratio in the experimental population.

Nanotechnology based approaches for detection and delivery of microRNA in healthcare and crop protection.

Nanobiotechnology has the potential to revolutionize diverse sectors including medicine, agriculture, food, textile and pharmaceuticals. Disease diagnostics, therapeutics and crop protection strategies are fast emerging using nanomaterials preferably nanobiomaterials. It has potential for development of novel nanobiomolecules which offer several advantages over conventional treatment methods. RNA nanoparticles with many unique features are promising candidates in disease treatment. The miRNAs are involved in many biochemical and developmental pathways and their regulation in plants and animals. These appear to be a powerful tool for controlling various pathological diseases in human, plants and animals, however there are challenges associated with miRNA based nanotechnology. Several advancements made in the field of miRNA therapeutics make it an attractive approach, but a lot more has to be explored in nanotechnology assisted miRNA therapy. The miRNA based technologies can be employed for detection and combating crop diseases as well. Despite these potential advantages, nanobiotechnology applications in the agricultural sector are still in its infancy and have not yet made its mark in comparison with healthcare sector. The review provides a platform to discuss nature, role and use of miRNAs in nanobiotechnology applications.