Reduced cold tolerance of viral-infected leafhoppers attenuates viral persistent epidemics.

Most arthropod-borne viruses produce intermittent epidemics in infected plants. However, the underlying mechanisms of these epidemics are unclear. Here, we demonstrated that rice stripe mosaic virus (RSMV), a viral pathogen, significantly increases the mortality of its overwintering vector, the leafhopper species Recilia dorsalis. Cold-stress assays indicated that RSMV reduces the cold tolerance of leafhoppers, a process associated with the downregulation of leafhopper cuticular protein genes. An RSMV-derived small RNA (vsiR-t00355379) was found to facilitate the downregulation of a leafhopper endocuticle gene that is mainly expressed in the abdomen (named RdABD-5) and is conserved across dipteran species. The downregulation of RdABD-5 expression in R. dorsalis resulted in fewer and thinner endocuticle lamellae, leading to decreased cold tolerance. This effect was correlated with a reduced incidence rate of RSMV in early-planted rice plants. These findings contribute to our understanding of the mechanism by which viral pathogens reduce cold tolerance in arthropod vectors and suggest an approach to managing the fluctuating prevalence of arboviruses. IMPORTANCE: Increasing arthropod vector dispersal rates have increased the susceptibility of crop to epidemic viral diseases. However, the incidence of some viral diseases fluctuates annually. In this study, we demonstrated that a rice virus reduces the cold tolerance of its leafhopper vector, Recilia dorsalis. This effect is linked to the virus-derived small RNA-mediated downregulation of a gene encoding a leafhopper abdominal endocuticle protein. Consequently, the altered structural composition of the abdominal endocuticle reduces the overwinter survival of leafhoppers, resulting in a lower incidence of RSMV infection in early-planted rice plants. Our findings illustrate the important roles of RNA interference in virus-vector insect-environment interactions and help explain the annual fluctuations of viral disease epidemics in rice fields.

Begomovirus Transmission to Tomato Plants Is Not Hampered by Plant Defenses Induced by Dicyphus hesperus Knight.

Plants can respond to insect infestation and virus infection by inducing plant defenses, generally mediated by phytohormones. Moreover, plant defenses alter host quality for insect vectors with consequences for the spread of viruses. In agricultural settings, other organisms commonly interact with plants, thereby inducing plant defenses that could affect plant-virus-vector interactions. For example, plant defenses induced by omnivorous insects can modulate insect behavior. This study focused on tomato yellow leaf curl virus (TYLCV), a plant virus of the family Geminiviridae and genus Begomovirus. It is transmitted in a persistent circulative manner by the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), posing a global threat to tomato production. Mirids (Hemiptera: Miridae) are effective biological control agents of B. tabaci, but there is a possibility that their omnivorous nature could also interfere with the process of virus transmission. To test this hypothesis, this study first addressed to what extent the mirid bug Dicyphus hesperus Knight induces plant defenses in tomato. Subsequently, the impact of this plant-omnivore interaction on the transmission of TYLCV was evaluated. Controlled cage experiments were performed in a greenhouse setting to evaluate the impact of mirids on virus transmission and vector acquisition by B. tabaci. While we observed a reduced number of whiteflies settling on plants exposed to D. hesperus, the plant defenses induced by the mirid bug did not affect TYLCV transmission and accumulation. Additionally, whiteflies were able to acquire comparable amounts of TYLCV on mirid-exposed plants and control plants. Overall, the induction of plant defenses by D. hesperus did not influence TYLCV transmission by whiteflies on tomato.

The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

IMPORTANCE: Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

A plant virus enhances odorant-binding protein 5 (OBP5) in the vector whitefly for more actively olfactory orientation to the host plant.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a notorious agricultural pest and the effective vector of many plant viruses worldwide. Cucurbit chlorotic yellows virus (CCYV), exclusively transmitted by B. tabaci in a semipersistent manner, is a serious causal agent in cucurbit crops in many countries. Plant viruses can manipulate the behaviors of insect vectors to promote the spread of themselves, but underlying mechanisms are remaining unclear. RESULTS: In this study, our observations indicated that B. tabaci, when carrying CCYV, oriented more actively to the host plant cucumber. Transcriptome analysis and quantitative polymerase chain reaction with reverse transcription analysis showed that the odorant-binding protein 5 (OBP5) was upregulated with viral acquisition. Sequence and phylogenetic analysis showed that BtabOBP5 was highly homologous with nine OBPs from other hemipteran insects. In addition, OBP5-silenced whiteflies significantly altered their orientation behavior towards cucumber plants and towards some typical volatile organic compounds released from cucumbers. CONCLUSION: This study described a novel mechanism by which the olfactory system of vector insects could be regulated by a semipersistent plant virus, thereby affecting insect olfactory behavior and relationship with host plants. These results provided a basis for developing potential olfaction-based pest management strategies in the future. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Key role of exportin 6 in exosome-mediated viral transmission from insect vectors to plants.

Exosomes play a key role in virus exocytosis and transmission. The exportin family is usually responsible for cargo nucleocytoplasmic trafficking, and they are frequently found in exosomes. However, the function of exportins sorted in exosomes remains unknown. Here, we successfully isolated "cup holder"-like exosomes from the saliva of ∼30,000 small brown planthoppers, which are vectors of rice stripe virus (RSV). RSV virions were packed in comparatively large exosomes. Four viral genomic RNAs at a certain ratio were identified in the saliva exosomes. The virions contained in the saliva exosomes were capable of replicating and causing disease in rice plants. Interference with each phase of the insect exosome system affected the transmission of RSV from the insect vectors to rice plants. Fragmented exportin 6 was coimmunoprecipitated with viral nucleocapsid protein in saliva and sorted to exosomes via interactions with the cargo sorting protein VPS37a. When the expression of exportin 6 was knocked down, the amounts of RSV secreted in saliva and rice plants were reduced by 60% and 74%, respectively. These results showed that exportin 6 acted as a vehicle for transporting RSV into exosomes to overcome the barrier of insect salivary glands for horizontal transmission. Exportin 6 would represent an ideal target that could be manipulated to control the outbreak of insect-borne viruses in the future.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

Flotillin 2 Facilitates the Infection of a Plant Virus in the Gut of Insect Vector.

Most plant viruses require insect vectors for transmission. One of the key steps for the transmission of persistent-circulative plant viruses is overcoming the gut barrier to enter epithelial cells. To date, little has been known about viral cofactors in gut epithelial cells of insect vectors. Here, we identified flotillin 2 as a plasma membrane protein that facilitates the infection of rice stripe virus (RSV) in its vector, the small brown planthopper. Flotillin 2 displayed a prominent plasma membrane location in midgut epithelial cells. The nucleocapsid protein of RSV and flotillin 2 colocalized on gut microvilli, and a nanomolar affinity existed between the two proteins. Knockout of flotillin 2 impeded the entry of virions into epithelial cells, resulting in a 57% reduction of RSV levels in planthoppers. The knockout of flotillin 2 decreased disease incidence in rice plants fed by viruliferous planthoppers from 40% to 11.7%. Furthermore, flotillin 2 mediated the infection of southern rice black-streaked dwarf virus in its vector, the white-backed planthopper. This work implies the potential of flotillin 2 as a target for controlling the transmission of rice stripe disease. IMPORTANCE Plant viral diseases are a major threat to world agriculture. The transmission of 80% of plant viruses requires vector insects, and 54% of vector-borne plant viruses are persistent-circulative viruses, which must overcome the barriers of gut cells with the help of proteins on the cell surface. Here, we identified flotillin 2 as a membrane protein that mediates the cell entry of rice stripe virus in its vector insect, small brown planthopper. Flotillin 2 displays a prominent cellular membrane location in midgut cells and can specifically bind to virions. The loss of flotillin 2 impedes the entry of virions into the midgut cells of vector insects and substantially suppresses viral transmission to rice. Therefore, flotillin 2 may be a promising target gene for manipulation in vector insects to control the transmission of rice stripe disease and perhaps that of other rice virus diseases in the future.

PEBP balances apoptosis and autophagy in whitefly upon arbovirus infection.

Apoptosis and autophagy are two common forms of programmed cell death (PCD) used by host organisms to fight against virus infection. PCD in arthropod vectors can be manipulated by arboviruses, leading to arbovirus-vector coexistence, although the underlying mechanism is largely unknown. In this study, we find that coat protein (CP) of an insect-borne plant virus TYLCV directly interacts with a phosphatidylethanolamine-binding protein (PEBP) in its vector whitefly to downregulate MAPK signaling cascade. As a result, apoptosis is activated in the whitefly increasing viral load. Simultaneously, the PEBP4-CP interaction releases ATG8, a hallmark of autophagy initiation, which reduces arbovirus levels. Furthermore, apoptosis-promoted virus amplification is prevented by agonist-induced autophagy, whereas the autophagy-suppressed virus load is unaffected by manipulating apoptosis, suggesting that the viral load is predominantly determined by autophagy rather than by apoptosis. Our results demonstrate that a mild intracellular immune response including balanced apoptosis and autophagy might facilitate arbovirus preservation within its whitefly insect vector.

Synergy between an emerging monopartite begomovirus and a DNA-B component.

In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

Diaphorina citri flavi-like virus localization, transmission, and association with Candidatus Liberibacter asiaticus in its psyllid host.

Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.

Alternative Splicing Landscape of Small Brown Planthopper and Different Response of JNK2 Isoforms to Rice Stripe Virus Infection.

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus, and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. IMPORTANCE Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus, which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.

A cytorhabdovirus-based expression vector in Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

The brown planthopper (BPH, Nilaparvata lugens), the small brown planthopper (SBPH, Laodelphax striatellus), and the white-backed planthopper (WBPH, Sogatella furcifera) are problematic insect pests and cause severe yield losses through phloem sap-sucking and virus transmission. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, has been developed as versatile expression platforms in SBPHs and cereal plants. However, bio-safe overexpression vectors based on recombinant BYSMV (rBYSMV) remain to be developed and applied to the three kinds of planthoppers. Here, we found that rBYSMV was able to infect SBPHs, BPHs and WBPHs through microinjection with crude extracts from rBYSMV-infected barley leaves. To ensure bio-safety of the rBYSMV vectors, we generated an rBYSMV mutant by deleting the accessory protein P3, a putative viral movement protein. As expected, the resulting mutant abolished viral systemic infection in barley plants but had no effects on BYSMV infectivity in insect vectors. Subsequently, we used the modified rBYSMV vector to overexpress iron transport peptide (ITP) in the three kinds of planthoppers and revealed the potential functions of ITP. Overall, our results provide bio-safe overexpression platforms to facilitate functional genomics studies of planthoppers.

Metatranscriptomic Sequencing Suggests the Presence of Novel RNA Viruses in Rice Transmitted by Brown Planthopper.

Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Differential expression of gut protein genes and population density of Arsenophonus contributes to sex-biased transmission of Bemisia tabaci vectored Cotton leaf curl virus.

Whitefly, Bemisia tabaci (Gennadius) is an important pest of cotton causing direct damage as sap feeder and vector of Cotton leaf curl virus (CLCuV). Previous few studies suggest that female whiteflies are more efficient vector of begomovirusthan males, however the sex-biased transmission efficiency is still not clearly understood. Present studies with B. tabaci AsiaII-1 haplotype showed higher virus transmission efficiency of females compared to males. This variable begomovirus transmission efficiency has been related to previously identifiedkey factors associated with B. tabaci. The higher density of endosymbiont Arsenophonus and variable expression of some midgut proteins genes i.e. Cyclophilin, Knottin, Hsp40, Hsp70 may be possibly imparting higher vector competency to the females compared to males. The present studies suggest low abundance of Arsenophonus spp. as well as lower expressionof Cyclophilin genein males as compared to females. This is further supplemented by overexpression of Knottin, Hsp40, and Hsp70 genes in males compared to females and thus collectively all these factors might be playing a key role in low virus transmission efficiency of males. The relative density of Arsenophonus spp. and expression of midgut proteins genes in male and female whitefly first time enriches our understanding about sex-biased transmission efficiency of begomovirus.

Transcriptome of the Maize Leafhopper (Dalbulus maidis) and Its Transcriptional Response to Maize Rayado Fino Virus (MRFV), Which It Transmits in a Persistent, Propagative Manner.

The corn leafhopper (Dalbulus maidis) is an important vector of maize rayado fino virus (MRFV), a positive-strand RNA (+ssRNA) marafivirus which it transmits in a persistent propagative manner. The interaction of D. maidis with MRFV, including infection of the insect and subsequent transmission to new plants, is not well understood at the molecular level. To examine the leafhopper-virus interaction, a D. maidis transcriptome was assembled and differences in transcript abundance between virus-exposed and naive D. maidis were examined at two time points (4 h and 7 days) post exposure to MRFV. The D. maidis transcriptome contained 56,116 transcripts generated from 1,727,369,026 100-nt paired-end reads from whole adult insects. The transcriptome of D. maidis shared highest identity and most orthologs with the leafhopper Graminella nigrifrons (65% of transcripts had matches with E values of <10-5) versus planthoppers Sogatella furcifera (with 23% of transcript matches below the E value cutoff) and Peregrinus maidis (with 21% transcript matches below the E value cutoff), as expected based on taxonomy. D. maidis expressed genes in the Toll, Imd, and Jak/Stat insect immune signaling pathways, RNA interference (RNAi) pathway genes, prophenoloxidase-activating system pathways, and immune recognition protein-encoding genes such as peptidoglycan recognition proteins (PGRPs), antimicrobial peptides, and other effectors. Statistical analysis (performed by R package DESeq2) identified 72 transcripts at 4 h and 67 at 7 days that were significantly responsive to MRFV exposure. Genes expected to be favorable for virus propagation, such as protein synthesis-related genes and genes encoding superoxide dismutase, were significantly upregulated after MRFV exposure. IMPORTANCE The transcriptome of the corn leafhopper, D. maidis, revealed conserved biochemical pathways for immunity and discovered transcripts responsive to MRFV-infected plants at two time points, providing a basis for functional identification of genes that either limit or promote the virus-vector interaction. Compared to other hopper species and the propagative plant viruses they transmit, D. maidis shared 15 responsive transcripts with S. furcifera (to southern rice black-streaked dwarf virus [SRBSDV]), one with G. nigrifrons (to maize fine streak virus [MFSV]), and one with P. maidis (to maize mosaic virus [MMV]), but no virus-responsive transcripts identified were shared among all four hopper vector species.

Survey and molecular detection of Sri Lankan cassava mosaic virus in Thailand.

Cassava plantations in an area of 458 hectares spanning five provinces along the Thailand-Cambodia border were surveyed from October 2018 to July 2019 to determine the prevalence of cassava mosaic disease (CMD) caused by Sri Lankan cassava mosaic virus (SLCMV) in the region. CMD prevalence was 40% in the whole area and 80% in Prachinburi, 43% in Sakaeo, 37% in Burium, 25% in Surin, and 19% in Sisaket provinces. Disease incidence of CMD was highest 43.08% in Sakaeo, followed by 26.78% in Prachinburi, 7% in Burium, 2.58% in Surin, and 1.25% in Sisaket provinces. Disease severity of CMD symptoms was mild chlorosis to moderate mosaic (2-3). The greatest disease severity was recorded in Prachinburi and Sakaeo provinces. Asymptomatic plants were identified in Surin (12%), Prachinburi (5%), Sakaeo (0.2%), and Buriram (0.1%) by PCR analysis. Cassava cultivars CMR-89 and Huai Bong 80 were susceptible to CMD. In 95% of cases, the infection was transmitted by whiteflies (Bemisia tabaci), which were abundant in Sakaeo, Buriram, and Prachinburi but were sparse in Surin; their densities were highest in May and June 2019. Nucleotide sequencing of the mitochondrial cytochrome oxidase 1 (mtCO1) gene of whiteflies in Thailand revealed that it was similar to the mtCO1 gene of Asia II 1 whitefly. Furthermore, the AV1 gene of SLCMV-which encodes the capsid protein-showed 90% nucleotide identity with SLCMV. Phylogenetic analysis of completed nucleotide sequences of DNA-A and DNA-B components of the SLCMV genome determined by rolling circle amplification (RCA) indicated that they were similar to the nucleotide sequence of SLCMV isolates from Thailand, Vietnam, and Cambodia. These results provide important insights into the distribution, impact, and spread of CMD and SLCMV in Thailand.

Comparative Transcriptome Analysis of Chemoreception Organs of Laodelphax striatellus in Response to Rice Stripe Virus Infection.

Many vector-borne viruses possess the ability to manipulate vector behaviors to facilitate their transmission. There is evidence that the mechanism of this phenomenon has been described in part as direct manipulation through regulating vector chemosensation. Rice stripe virus (RSV) is transmitted by the small brown planthopper, Laodelphax striatellus (Fallen), in a persistent, circulative-propagative manner. The effect of RSV infection on the olfactory system of L. striatellus has not been fully elucidated. Here, we employed transcriptomic sequencing to analyze gene expression profiles in antennae, legs and heads (without antennae) from L. striatellus females and males with/without RSV infection. Comparisons of the differentially expressed genes (DEGs) among antennae, legs and heads indicated that tissue-specific changes in the gene expression profile were greater than sex-specific changes. A total of 17 olfactory related genes were differentially expressed in viruliferous antennae as compared to nonviruliferous antennae, including LstrOBP4/9, LstrCSP1/2/5, LstrGR28a/43a/43a-1, LstrIR1/2/NMDA1, LstrOR67/85e/56a/94 and LstrSNMP2/2-2. There are 23 olfactory related DEGs between viruliferous and nonviruliferous legs, including LstrOBP2/3/4/12/13, LstrCSP13/5/10, LstrIR1/2/Delta2/Delta2-1/kainate2/NMDA2, LstrOR12/21/31/68 and LstrORco. A low number of olfactory related DEGs were found between viruliferous and nonviruliferous heads, including LstrCSP1, LstrOBP2, LstrOR67 and LstrSNMP2-2. Among these DEGs, the expression patterns of LstrOBP2, LstrOBP3 and LstrOBP9 in three tissues was validated by quantitative real-time PCR. The demonstration of overall changes in the genes in L. striatellus' chemoreception organs in response to RSV infection would not only improve our understanding of the effect of RSV on the olfactory related genes of insect vectors but also provide insights into developing approaches to control the plant virus transmission and spread as well as pest management in the future.

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.

Impacts of Cucurbit Chlorotic Yellows Virus (CCYV) on Biological Characteristics of Its Vector Bemisia tabaci (Hemiptera: Aleyrodidae) MED Species.

Plant viruses can change the phenotypes and defense pathways of the host plants and the performance of their vectors to facilitate their transmission. Cucurbit chlorotic yellows virus (CCYV) (Crinivirus), a newly reported virus occurring on cucurbit plants and many other plant species, is transmitted specifically by Bemisia tabaci MEAM1 (B biotype) and MED (Q biotype) cryptic species in a semipersistent manner. This study evaluated the impacts of CCYV on B. tabaci to better understand the plant-virus-vector interactions. By using CCYV-B. tabaci MED-cucumber as the model, we investigated whether or how a semipersistent plant virus impacts the biology of its whitefly vector. CCYV mRNAs were detectable in nymphs from first to fourth instars and adults of B. tabaci with different titers. Nymph instar durations and adult longevity of female whiteflies greatly extended on CCYV-infected plants, but nymph instar durations and adult longevity of male whiteflies were not significantly influenced. In addition, the body length and oviposition increased in adults feeding on CCYV-infected plants, but the hatching rates of eggs and survival rates of different stages were not affected. Most interestingly, the sex ratio (male:female) significantly reduced to 0.5:1 in whitefly populations on CCYV-infected plants, while the ratio remained about 1:1 on healthy plants. These results indicated that CCYV can significantly impact the biological characteristics of its vector B. tabaci. It is speculated that CCYV and B. tabaci have established a typical mutualist relationship mediated by host plants.