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.

Association of a novel begomovirus species with fenugreek yellow vein disease in India.

BACKGROUND: Fenugreek (Trigonella foenum-graecum L.) is an annual medicinal and spice crop belonging to the family Fabaceae. The occurrence of a yellow vein disease was recorded in fenugreek in Jodhpur (India) in 2022. The infection of begomoviruses in legume crops results in significant yield loss and major economic loss. The current study reports an association of a novel begomovirus species associated with yellow vein disease in Fenugreek. METHODS AND RESULTS: In symptomatic fenugreek plants, geminivirus-like particles were visible under a transmission electron microscope. Further, nucleotide sequence analysis of the rolling circle amplified product revealed 2743 nucleotide DNA-A genome with close relatedness to French bean leaf curl virus (88.21%) and Senna leaf curl virus (87.63%). It was proposed as a new begomovirus species, Fenugreek yellow vein Rajasthan virus. The genome organization suggested the presence of a typical nonanucleotide sequence along with 7 ORFs in DNA-A. A possible recombination event took place in the coat protein (V1) region with Pedilanthus leaf curl virus and Chilli leaf curl virus as major and minor parents. The recombinant virus poses possible threats to several other legume crops. To the best of our knowledge, this is the first report of the association of FeYVRaV with fenugreek yellow vein disease from northwestern India. CONCLUSIONS: In conclusion, the presence of a novel begomovirus species associated with yellow vein disease in fenugreek is alarming and needs further studies on its infectivity to prevent its spread to legume crops.

Diversity of transgenes in sustainable management of insect pests.

Insecticidal transgenes, when incorporated and expressed in plants, confer resistance against insects by producing several products having insecticidal properties. Protease inhibitors, lectins, amylase inhibitors, and chitinase genes are associated with the natural defenses developed by plants to counter insect attacks. Several toxin genes are also derived from spiders and scorpions for protection against insects. Bacillus thuringiensis Berliner is a microbial source of insecticidal toxins. Several methods have facilitated the large-scale production of transgenic plants. Bt-derived cry, cyt, vip, and sip genes, plant-derived genes such as lectins, protease inhibitors, and alpha-amylase inhibitors, insect cell wall-degrading enzymes like chitinase and some proteins like arcelins, plant defensins, and ribosome-inactivating proteins have been successfully utilized to impart resistance to insects. Besides, transgenic plants expressing double-stranded RNA have been developed with enhanced resistance. However, the long-term effects of transgenes on insect resistance, the environment, and human health must be thoroughly investigated before they are made available for commercial planting. In this chapter, the present status, prospects, and future scope of transgenes for insect pest management have been summarized and discussed.

Delivery of progeny virus from the infectious clone of cucumber green mottle mosaic virus and quantification of the viral load in different host plants.

Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is a widely occurring tobamovirus in cucurbits. The genome of CGMMV has been used previously for the expression of foreign genes in the plant. High throughput delivery and high viral titer are important requirements of foreign protein expression in plant through virus genome-based vector, in this study, Agrobacterium containing infectious construct of CGMMV was infiltrated through syringe, vacuum and high-speed spray to N. benthamiana, cucumber and bottle gourd leaves. The success rate of systemic infection of CGMMV agro-construct through all three methods was higher (80-100%) in N. benthamiana compared to the cucurbits (40-73.3%). To determine the high-throughput delivery of CGMMV in the plant system, four delivery methods viz. rubbing, syringe infiltration, vacuum infiltration and high-speed spray using the progeny virus derived through CGMMV agro-construct were compared in the three different plant species. Based on the rate of systemic infection and time required to perform delivery by different methods, vacuum infiltration was found most efficient for the high-throughput delivery of CGMMV. The quantification of CGMMV through qPCR revealed that CGMMV load varied considerably in leaf and fruit tissues depending with the time of infection. Immediately after expression of symptoms, a high load of CGMMV (~ 1 µg/100 mg of tissues) was noticed in young leaves of N. benthamiana and cucumber. In bottle gourd leaves, the CGMMV load was far low compared to N. benthamiana and cucumber plants. In the fruit tissues of cucumber and bottle gourd higher virus load was observed in mature fruit but not in immature fruit. The findings of the present study will serve as an important base line information to produce foreign protein through CGMMV genome-vector. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03630-y.

Mitochondrial genetic diversity of Thrips tabaci (Thysanoptera: Thripidae) in onion growing regions of the United States.

Onion thrips (Thrips tabaci Lindeman, Thysanoptera: Thripidae) causes severe damage to many horticultural and agronomic crops worldwide. It also acts as a vector of several plant viruses. T. tabaci is a key pest of Allium cepa in the United States. However, there is limited information available on the genetic variation within and between T. tabaci populations in the United States and its key evolutionary parameters. In the current study, 83 T. tabaci specimens were collected from A. cepa from 15 different locations comprising four states of the United States. A total of 92 mtCOI gene sequences of T. tabaci from A. cepa were analyzed to understand the genetic diversity and structure of T. tabaci collected from onion host. Seven distinct haplotypes of T. tabaci infesting A. cepa were identified from the current collection, while nine T. tabaci sequences retrieved from GenBank comprised 5 haplotypes. Overall, 15 haplotypes of T. tabaci infesting A. cepa were identified in the world that includes the ten haplotypes in the United States. In the phylogenetic analysis, all the populations collected during the study clustered with thelytokous lineage, while T. tabaci sequences retrieved from GenBank corresponded to leek-associated arrhenotokous lineage. The highest genetic variation was found in Elba and Malheur populations with 3 haplotypes identified in each. The results suggest that haplotypes 1 and 7 are more frequently prevailing haplotypes in the north-western United States, with haplotype 1 being the predominant all over the country. The eastern United States appears to have a more diverse group of haplotypes. The populations from Hungary constituted distinct haplotypes and a haplotype from Kingston linked it with the predominant haplotype.

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.

Silencing of Thrips palmi UHRF1BP1 and PFAS Using Antisense Oligos Induces Mortality and Reduces Tospovirus Titer in Its Vector

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, legumes, and ornamentals. In addition, it transmits several plant viruses. T. palmi genes associated with innate immunity, endocytosis-related pathways, and cuticular development are highly enriched in response to Groundnut bud necrosis orthotospovirus (GBNV, genus Orthotospovirus, family Tospoviridae) infection. As the previous transcriptomic study suggested the involvement of T. palmi UHRF1BP1 and PFAS in GBNV infection, these two genes were targeted for silencing using antisense oligonucleotides (ASOs), and the effects on thrips' fitness and virus acquisition were observed. Phosphorothioate modification of ASOs was carried out by replacing the nonbridging oxygen atom with a sulfur atom at the 3' position to increase nuclease stability. The modified ASOs were delivered orally through an artificial diet. Exposure to ASOs reduced the target mRNA expression up to 2.70-fold optimally. Silencing of T. palmi UHRF1BP1 and PFAS induced 93.33% mortality that further increased up to 100% with an increase in exposure. Silencing of T. palmi UHRF1BP1 and PFAS also produced morphological deformities in the treated T. palmi. GBNV titer in T. palmi significantly declined post-exposure to ASOs. This is the first-ever report of silencing T. palmi UHRF1BP1 and PFAS using modified ASO to induce mortality and impair virus transmission in T. palmi. T. palmi UHRF1BP1 and PFAS would be novel genetic targets to manage thrips and restrict the spread of tospovirus.

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.

Transcriptomic Changes of Bemisia tabaci Asia II 1 Induced by Chilli Leaf Curl Virus Trigger Infection and Circulation in Its Vector.

Bemisia tabaci (Hemiptera: Aleyrodidae) is a highly efficient vector in the spread of chilli leaf curl virus (ChiLCV, Begomovirus) which is a major constraint in the production of chilli in South Asia. Transcriptome analysis of B. tabaci post-6 h acquisition of ChiLCV showed differential expression of 80 (29 upregulated and 51 downregulated) genes. The maximum number of DEGs are categorized under the biological processes category followed by cellular components and molecular functions. KEGG analysis of DEGs showed that the genes are involved in the functions like metabolism, signaling pathways, cellular processes, and organismal systems. The expression of highly expressed 20 genes post-ChiLCV acquisition was validated in RT-qPCR. DEGs such as cytosolic carboxypeptidase 3, dual-specificity protein phosphatase 10, 15, dynein axonemal heavy chain 17, fasciclin 2, inhibin beta chain, replication factor A protein 1, and Tob1 were found enriched and favored the virus infection and circulation in B. tabaci. The present study provides an improved understanding of the networks of molecular interactions between B. tabaci and ChiLCV. The candidate genes of B. tabaci involved in ChiLCV transmission would be novel targets for the management of the B. tabaci-begomovirus complex.

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.

Topical Spray of dsRNA Induces Mortality and Inhibits Chilli Leaf Curl Virus Transmission by Bemisia tabaci Asia II 1.

Chilli leaf curl virus (ChiLCV; genus: Begomovirus), transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a persistent-circulative manner, is a major constraint in chilli production. The present study demonstrates for the first time that a topical spray of naked double-stranded RNA (dsRNA) on chilli plants causes mortality and inability to acquire and transmit ChiLCV in B. tabaci. dsRNA targeting heat shock protein 70 (hsp70) and fasciclin 2 (fas2) of B. tabaci Asia II 1 was first assessed under controlled conditions through oral delivery. Hsp70 and fas2 dsRNA resulted in up to 82.22% and 72% mortality of B. tabaci and around 12.4- and 8.5-fold decreases in mRNA levels, respectively, 24 h post-ingestion. ChiLCV copies in hsp70 dsRNA-fed B. tabaci steadily decreased with an increase in dsRNA concentration and were undetectable at a higher concentration of dsRNA. However, ChiLCV copies significantly increased in fas2 dsRNA-fed B. tabaci. Transmission of ChiLCV by B. tabaci was completely inhibited post-24 h feeding on hsp70 dsRNA at 3 µg/mL. Naked hsp70 dsRNA was topically sprayed on ChiLCV-infected chilli plants like an insecticide. 67.77% mortality of B. tabaci, 4.6-fold downregulation of hsp70 mRNA, and 1.34 × 1015-fold decreased ChiLCV copies in B. tabaci were recorded when adults were exposed to the dsRNA-treated plants under semi-field conditions. Foliar application of naked dsRNA reduced the ChiLCV transmission by 75% without any visible symptoms in the inoculated plants. A total of 2 consecutive sprays of dsRNA provided significant protection to B. tabaci for up to 20 days under semi-field conditions.

Transovarial Transmission of Dolichos Yellow Mosaic Virus by Its Vector, Bemisia tabaci Asia II 1.

The cultivation of dolichos bean [Lablab purpureus (L.) Sweet] has been severely affected by dolichos yellow mosaic virus (DoYMV, Begomovirus) transmitted by whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). We tested the transovarial transmission of DoYMV in next-generation B. tabaci by PCR, real-time PCR, Southern blot hybridization, and biological transmission. The eggs, laid by DoYMV-exposed B. tabaci, carry the virus in a unique pattern. Only the eggs laid in between 3 and 6 days post virus acquisition by a parent B. tabaci were DoYMV positive. When tested individually in real-time PCR, around 31-53% of the eggs carried the virus. The presence of DoYMV in ovaries and F1 eggs was further substantiated by the hybridization of a Cy3-conjugated nucleic acid probe complementary to the viral strand of DoYMV. Viral DNA was also detected in F1 adults and F2 eggs. B. tabaci progenies carried not only the DoYMV DNA but were also infective. The F1 adults transmitted DoYMV to all tested plants and produced strong yellow mosaic symptoms. An increase in viral copies from egg to nymphal stage indicated propagation of DoYMV in B. tabaci. However, the increase was for a short period and decreased thereafter. The present study provides the first evidence of transovarial transmission and propagation of a bipartite begomovirus in its vector, B. tabaci Asia II 1. The transovarial transmission and replication of DoYMV in B. tabaci have great epidemiological relevance as B. tabaci can serve as a major host of the virus to bridge the gap between the cropping seasons.

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.

How many begomovirus copies are acquired and inoculated by its vector, whitefly (Bemisia tabaci) during feeding?

Begomoviruses are transmitted by whitefly (Bemisia tabaci Gennadius, Hemiptera: Aleyrodidae) in a persistent-circulative way. Once B. tabaci becomes viruliferous, it remains so throughout its life span. Not much is known about the copies of begomoviruses ingested and/or released by B. tabaci during the process of feeding. The present study reports the absolute quantification of two different begomoviruses viz. tomato leaf curl New Delhi virus (ToLCNDV, bipartite) and chilli leaf curl virus (ChiLCV, monopartite) at different exposure of active acquisition and inoculation feeding using a detached leaf assay. A million copies of both the begomoviruses were acquired by a single B. tabaci with only 5 min of active feeding and virus copy number increased in a logarithmic model with feeding exposure. Whereas, a single B. tabaci could inoculate 8.21E+09 and 4.19E+11 copies of ToLCNDV and ChiLCV, respectively in detached leaves by 5 min of active feeding. Virus copies in inoculated leaves increased with an increase in feeding duration. Comparative dynamics of these two begomoviruses indicated that B. tabaci adult acquired around 14-fold higher copies of ChiLCV than ToLCNDV 24 hrs post feeding. Whereas, the rate of inoculation of ToLCNDV by individual B. tabaci was significantly higher than ChiLCV. The study provides a better understanding of begomovirus acquisition and inoculation dynamics by individual B. tabaci and would facilitate research on virus-vector epidemiology and screening host resistance.

Effect of Neonicotinoids on Bacterial Symbionts and Insecticide-Resistant Gene in Whitefly, Bemisia tabaci.

The silverleaf whitefly, Bemisia tabaci (Gennadius, Hemiptera: Aleyrodidae), is a major threat to field and horticultural crops worldwide. Persistent use of insecticides for the management of this pest is a lingering problem. In the present study, the status of sensitivity of B. tabaci to two neonicotinoids, imidacloprid and thiamethoxam, was evaluated. The expression pattern of two cytochrome P450 (cyp) genes and changes in the relative amount of symbionts in insecticide-treated B. tabaci were also assessed. Quantitative PCR (qPCR) studies indicate that the CYP6CM1 and CYP6CX1 genes were always expressed higher in imidacloprid-treated whitefly, suggesting a correlation between gene expression and the insect's ability to detoxify toxic compounds such as insecticides. In addition, the thiamethoxam-treated population harbored higher Portiera and lower Rickettsia titers, whereas the imidacloprid-treated population harbored more Rickettsia at different time intervals. Interestingly, we also examined that an increase in exposure to both the insecticides resulted in a reduction in the mutualistic partners from their insect host. These differential responses of endosymbionts to insecticide exposure imply the complex interactions among the symbionts inside the host insect. The results also provide a deeper understanding of the molecular mechanism of resistance development that might be useful for formulating effective management strategies to control B. tabaci by manipulating symbionts and detoxifying genes.

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi.

Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips-tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips-tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

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.

Simulation of leaf curl disease dynamics in chili for strategic management options.

Leaf curl, a whitefly-borne begomovirus disease, is the cause of frequent epidemic in chili. In the present study, transmission parameters involved in tripartite interaction are estimated to simulate disease dynamics in a population dynamics model framework. Epidemic is characterized by a rapid conversion rate of healthy host population into infectious type. Infection rate as basic reproduction number, R0 = 13.54, has indicated a high rate of virus transmission. Equilibrium population of infectious host and viruliferous vector are observed to be sensitive to the immigration parameter. A small increase in immigration rate of viruliferous vector increased the population of both infectious host and viruliferous vector. Migrant viruliferous vectors, acquisition, and transmission rates as major parameters in the model indicate leaf curl epidemic is predominantly a vector -mediated process. Based on underlying principles of temperature influence on vector population abundance and transmission parameters, spatio-temporal pattern of disease risk predicted is noted to correspond with leaf curl distribution pattern in India. Temperature in the range of 15-35 °C plays an important role in epidemic as both vector population and virus transmission are influenced by temperature. Assessment of leaf curl dynamics would be a useful guide to crop planning and evolution of efficient management strategies.

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.