High Throughput Sequencing-Aided Survey Reveals Widespread Mixed Infections of Whitefly-Transmitted Viruses in Cucurbits in Georgia, USA.

Viruses transmitted by the sweet potato whitefly (Bemisia tabaci) have been detrimental to the sustainable production of cucurbits in the southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Symptomatic samples were collected and small RNA libraries were prepared and sequenced from three cantaloupes, four cucumbers, and two yellow squash samples. An analysis of the sequences revealed the presence of the criniviruses cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and the begomovirus cucurbit leaf crumple virus (CuLCrV). CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples (n = 820) tested. The level of mixed infections was high in all the cucurbits, with most plants tested being infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV, were assembled from the small RNA sequences. An analysis of the coding regions showed low genetic variability among isolates from different hosts. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and USA isolates. This work enhances our understanding of the distribution of viruses on cucurbits in South Georgia and will be useful to develop strategies for managing the complex of whitefly-transmitted viruses in the region.

Molecular insights on potato yellow vein crinivirus infections in the highlands of Colombia.

Potato yellow vein virus (PYVV) was detected in potatoes grown in the Central highlands, north of Bogotá (~3000 m altitude), Colombia. At this altitude viral whitefly vectors are largely absent, but infection persists because of the use of uncertified tubers. Plants with typical PYVV-induced yellowing symptoms, as well as with atypical yellowing or non-symptomatic symptoms were sampled at three separate geographical locations. PYVV presence was assessed by RT-PCR, and several plants were subjected to high-throughput sequencing (HTS) of their small RNA (sRNA) populations. Complete or almost complete sequences of four PYVV isolates were thus reconstructed, all from symptomatic plants. Three viral isolates infected plants singly, while the fourth co-infected the plant together with a potyvirus. Relative proportions of sRNAs to each of the three crinivirus genomic RNAs were found to remain comparable among the four infections. Genomic regions were identified as hotspots of sRNA formation, or as regions that poorly induced sRNAs. Furthermore, PYVV titres in the mixed versus single infections remained comparable, indicating an absence of synergistic/antagonistic effects of the potyvirus on the accumulation of PYVV. Daughter plants raised in the greenhouse from tubers of the infected, field-sampled plants displayed mild PYVV infection symptoms that disappeared with time, demonstrating the occurrence of recovery and asymptomatic infection phenotypes in this pathosystem.

A rapid fluorescence-based real-time isothermal assay for the detection of Cucurbit yellow stunting disorder virus in squash and watermelon plants.

Cucurbit yellow stunting disorder virus (CYSDV) is a single-stranded positive-sense RNA virus that produces devastating disease in watermelon and squash. Foliar symptoms of CYSDV consist of interveinal yellowing, brittleness, and thickening of older leaves leading to reduced plant vigor. A rapid diagnostic method for CYSDV would facilitate early detection and implementation of best viral-based management practices. We developed a rapid isothermal reverse transcription-recombination polymerase amplification (exo RT-RPA) assay for the detection of CYSDV. The primers and a 6-fluorescein amidite (6-FAM) probe were developed to target the nucleocapsid gene. The real-time assay detected CYSDV at 2.5 pg purified total RNA extracted from CYSDV-infected leaf tissue and corresponded to 10 copies of the target molecule. The assay was specific and did not cross-react with other common cucurbit viruses found in Florida and Georgia. The performance of the exo RT-RPA was evaluated using crude extract from 21 cucurbit field samples and demonstrated that the exo RT-RPA is a rapid procedure, thus providing a promising novel alternative approach for the detection of CYSDV.

Outbreaks of Bemisia tabaci Mediterranean species in vegetable crops in São Paulo and Paraná States, Brazil.

The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most important agricultural pests and virus vectors worldwide. Bemisia tabaci is considered a complex of cryptic species with at least 44 species. Among them, the species Middle East-Asia Minor 1 (MEAM1, formerly B biotype) and Mediterranean (MED, formerly Q biotype) are the most important, and they have attained global status. In Brazil, MEAM1 was first reported in the 1990s and is currently the predominant species in the country, meanwhile, MED was recently reported in the South and Southeast regions and was found to be mainly associated with ornamental plants. Currently, an increasing problem in the management of whitefly infestations in greenhouses associated with bell pepper was observed in São Paulo State, Brazil. The whiteflies were collected and identified based on a microsatellite locus (primer pair BEM23F and BEM23R) and the mitochondrial cytochrome oxidase I gene followed by restriction fragment length polymorphism analysis and sequencing. We observed that MED was the predominant species collected on bell pepper, but it was also found on tomato, cucumber, eggplant, and weeds grown in greenhouses. In open field, we found MED on tomatoes, bell peppers, and eggplants. In addition, MED was identified in Goiás State in association with ornamental plants. The begomovirus Tomato severe rugose virus and the crinivirus Tomato chlorosis virus was detected on bell pepper and tomato, respectively. Only MED specimens were found associated with the virus-infected plants. Moreover, we also investigated the endosymbionts present in the MED whiteflies. The collected populations of B. tabaci MED harbored a diversity of secondary endosymbionts, with Hamiltonella (H) found predominantly in 89 specimens of the 129 tested. These results represent a new concern for Brazilian agriculture, especially for the management of the newly introduced whitefly MED species, which must be implemented to limit the spreading and establishment of this pest in different crops in this country.

Lettuce Chlorosis Virus Disease: A New Threat to Cannabis Production.

In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85-99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisiatabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected 'healthy' cannabis, Lactucasativa, and Catharanthusroseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

Prevalence, epidemiology and molecular studies of Tomato chlorosis virus (ToCV) in South Africa.

Criniviruses accumulate in the phloem tissue and damage crops by reducing chlorophyll which is essential for plant growth and development. Tomato chlorosis crinivirus (ToCV) is vectored by several whitefly species that damage tomato crops throughout the world. In South Africa, ToCV is a poorly studied pathogen of global economic importance. Therefore, a national survey was initiated to investigate the occurrence and distribution of criniviruses infecting tomato crops in South Africa. Whitefly infested tomato crops exhibiting interveinal leaf chlorosis and chlorotic flecking symptoms were assayed for crinivirus infections using a multiplex reverse transcription polymerase reaction (RT-PCR) approach to assess for the presence of crinivirus species that are known to infect solanaceous hosts. Next-generation sequencing (NGS) was used to generate the complete genome of ToCV from South Africa. Results from the survey indicated that ToCV is presently the only crinivirus species infecting tomatoes in South Africa. Blast analysis showed that the RNA-1 segment of ToCV from South Africa (ToCR1-186) matched 99% to Spanish isolates. On the other hand, the RNA-2 (ToCR2-186) segment matched 98% to a South Korean isolate and three Spanish isolates. Although recombination events were not detected, phylogenetic studies showed inconsistencies in the grouping of RNA-1 and RNA-2 segments for some of the ToCV isolates analyzed in this study. Therefore, we suggest the possibility of intraspecific reassortment. This is the first comprehensive study and full genome sequence of ToCV from South Africa. The information generated from this study is intended to raise awareness of ToCV infections on tomato crops in South Africa.

An isolate of sweet potato chlorotic stunt virus from Brazil with a distinct genome organization.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae), is an economically important pathogen of sweet potato. In the present work, the nucleotide sequences of two RNA segments of SPCSV (isolate SPCSV-UNB-01) were determined by MiSeq Illumina sequencing of samples of sweet potato plants grafted onto Ipomoea setosa. A comparative analysis of the genome organization of SPCSV-UNB-01 and other SPCSV sequences showed that RNA1 was lacking p22, and p5.1 and that p5.2. was absent in RNA2, indicating a unique genomic pattern. SPCSV-UNB-01 contained longer p6 and p5 regions, with little similarity to orthologous sequences. Sequence comparison did not reveal any previously identified functional domains within these open reading frames (ORFs). No recombination or rearrangement events were detected. Phylogenetic analysis suggested the possibility of separate entries of SPCSV into South America based on the genetic distance between SPCSV-UNB-01 and the Peruvian isolate m2-47. Samples from northeastern Brazil (State of Pernambuco) were positive for SPCSV when tested using specific primers for the major coat protein (CP) gene. This is the first full-length genome sequence of SPCSV-UNB-01 from Brazil.

Development of a graft inoculation method and a real-time RT-PCR assay for monitoring Tomato chlorosis virus infection in tomato.

A graft inoculation method coupled with RT-qPCR was developed for monitoring ToCV infection in tomato plants. Ten seed-grown tomato seedlings were graft inoculated with phloem tissue-containing stem segments from a ToCV-infected tomato plants. Another group of tomato seedling were grafted with similar stem segments from a healthy tomato plant as mock inoculated control. The CP gene of ToCV was cloned under the control of T7 promoter and in vitro synthesized RNA was used as a standard for quantification. Total RNA was isolated from leaf samples of ToCV-inoculated and mock-inoculated control plants before the inoculation and 1-60 days post inoculation (dpi). The presence and the titer of ToCV were determined from all ToCV-inoculated or mock-inoculated control plants by RT-qPCR. After 15 dpi, ToCV was detected in 20-30% of graft-inoculated plants. The infection rate then increased progressively and reached to 70-80% by 60 dpi. Titer of ToCV was at the detectable level at 15 dpi and increased and reached to maximum level by 40 dpi and then started to decrease. The results showed that patch grafting is a simple and efficient method for experimental inoculation of ToCV and can be used as an alternative and/or complementary to vector transmission in the laboratories. The patch grafting could be combined with RT-qPCR and used for infecting and quantitative monitoring of ToCV or other phloem-limited viruses in tomato or in other plants.

Complete genome sequence of a lettuce chlorosis virus isolate from China and genome recombination/rearrangement analysis.

We determined the complete genome sequence of a lettuce chlorosis crinivirus (LCV) from China (LCV-NJ). The bipartite genome of LCV-NJ consists of RNA1 and RNA2 which are 8165 and 8454 nucleotides (nt) in length, respectively. The genomic structure of LCV-NJ RNA1 resembles that of LCV-California, an isolate with four open reading frames (ORFs) in RNA1. Although the amino acid sequences of ORF 1a and 1b have 92 and 99% identity between LCV-NJ and LCV-California, ORF 2 and ORF3 of LCV-NJ share only 63 and 71% identity with those of LCV-California, respectively. In addition LCV-NJ RNA2 contains 9 ORFs, compared to 10 ORFs in LCV-California. ORF10 was missing due to the deletion of a 173-nt sequence within the 3'-terminal region of LCV-NJ RNA2. Insertion or deletion of sequences of varying lengths was also observed in RNA1 and other regions of RNA2. Based on these findings, we propose that LCV-NJ/LCV-California may have undergone genome recombination and/or rearrangement in RNA1 and RNA2.

Molecular Characterization and Detection of a Genetically Distinct Tomato Chlorosis Virus Strain in Taiwan.

The whitefly-transmitted tomato chlorosis virus (ToCV) belonging to the genus Crinivirus (family Closteroviridae) affects tomato production worldwide. ToCV was first recorded in Taiwan in 1998 affecting tomato production. In this study, a local virus isolate XS was obtained, after serial whitefly transmissions from a diseased tomato plant displaying general chlorosis were collected in central Taiwan. The whole genome sequence of XS was determined from cDNA fragments amplified by reverse transcription (RT)-PCR, first using the degenerate primers for viruses of Closteroviridae and followed by degenerate and specific primers designed on available sequences of the ToCV isolates. The nucleotide (nt) sequences of RNA-1 and RNA-2 of the XS shared low identities of 77.8 to 78% and 78 to 78.1%, respectively, with genome segments of other ToCV isolates. Nevertheless, the viral RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (Hsp70h), and major capsid protein (CP) shared 88.3 to 96.2% amino acid (aa) identities with other ToCV isolates, indicating that XS is a new strain of this virus. Phylogenetic analyses of these three proteins indicated that all ToCV isolates from different counties outside Taiwan are closely related and clustered in the same clade, whereas the XS isolate is distinct and forms a unique branch. A one tube RT-PCR assay using primers designed from the genomic sequence of the XS was able to detect the ToCV-XS in infected tomato plants and in individual whiteflies. A field survey during 2013 to 2016 revealed a high ToCV-XS prevalence of 60.5% in 172 tested tomato samples, demonstrating that ToCV-XS is becoming an emerging threat for tomato production in Taiwan.

Detection and epidemic dynamic of ToCV and CCYV with Bemisia tabaci and weed in Hainan of China.

BACKGROUND: In recent years, two of the crinivirus, Tomato chlorosis virus (ToCV) and Cucurbit chlorotic yellows virus (CCYV) have gained increasing attention due to their rapid spread and devastating impacts on vegetable production worldwide. Both of these viruses are transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), in a semi-persistent manner. Up to now, there is still lack of report in Hainan, the south of China. METHODS: We used observational and experimental methods to explore the prevalence and incidence dynamic of CCYV and ToCV transmitted by whiteflies in Hainan of China. RESULTS: In 2016, the chlorosis symptom was observed in the tomato and cucumber plants with a large number of B. tabaci on the infected leaves in Hainan, China, with the incidence rate of 69.8% and 62.6% on tomato and cucumber, respectively. Based on molecular identification, Q biotype was determined with a viruliferous rate of 65.0% and 55.0% on the tomato and cucumber plants, respectively. The weed, Alternanthera philoxeroides near the tomato and cucumber was co-infected by the two viruses. Furthermore, incidence dynamic of ToCV and CCYV showed a close relationship with the weed, Alternanthera philoxeroides, which is widely distributed in Hainan. CONCLUSION: Our results firstly reveal that the weed, A. philoxeroides is infected by both ToCV and CCYV. Besides, whiteflies showed a high viruliferous rate of ToCV and CCYV. Hainan is an extremely important vegetable production and seed breeding center in China. If the whitefly can carry these two viruses concurrently, co-infection in their mutual host plants can lead to devastating losses in the near future.

Emergence and epidemiology of Cucurbit yellow stunting disorder virus in the American Desert Southwest, and development of host plant resistance in melon.

Cucurbit yellow stunting disorder virus (CYSDV), emerged in the Sonoran Desert region of the southwestern USA in 2006 and has become well established. Symptoms induced by CYSDV infection include a striking interveinal chlorosis or yellowing and reduced yield and quality. The virus is transmitted by Bemisia tabaci, and the cryptic species MEAM1 has been present in the region since the early 1990s. CYSDV has now become the most economically important of the viruses affecting cucurbit production in the southwestern US. Here, we present a review of recent studies on CYSDV in the southwestern US, with implications for management of this virus throughout the world. Field surveys have established that CYSDV results in late-season infection of spring melon crops with limited economic impact; however, all summer and fall cucurbits become infected shortly after emergence due to high B. tabaci populations and abundant sources of inoculum. Studies have also demonstrated that CYSDV has an extensive host range among crops and weeds prevalent in the region. Recent studies demonstrated considerable variation in virus accumulation and transmission rates among the host plants evaluated as potential reservoirs. Cucurbit hosts had the highest CYSDV titers, were efficient sources for virus acquisition, and showed a positive correlation between titer in source plants and transmission to cucurbit plants. Non-cucurbit hosts had significantly lower CYSDV titers and varied in their capacity to serve as sources for transmission. Experiments demonstrated that multiple factors influence the efficiency with which a host plant species will be a reservoir for vector transmission of CYSDV to crops. Melon PI 313970 was identified as a new source of host plant resistance to CYSDV, in addition to the previously identified TGR 1551 (=PI 482420) and TGR 1937 (=PI 482431). Potential new sources of CYSDV resistance were identified by field screening of ca. 500 melon accessions with naturally occurring inoculum from 2007 through 2012. Host plant resistance to B. tabaci has also been identified in melon germplasm resistant to CYSDV and could be an important factor in reducing losses to CYSDV. Resistance to CYSDV is being transferred to US western shipping type cantaloupe and honeydew.

Field evaluation of Cucurbit yellow stunting disorder virus transmission by Bemisia tabaci.

Cucurbit yellow stunting disorder virus (CYSDV) is a whitefly-transmitted Crinivirus (Closteroviridae) that impacts melon production in many parts of the world including the USA. It has been responsible for melon crop loss in the southwestern U.S. since 2006 when it was first identified. Control strategies have revolved mainly around chemical control, but research to identify suitable products and approaches to implementing them have lagged. The current study investigated the performance of four systemic insecticides in the field while concurrently tracking CYSDV disease progression after controlled and natural whitefly inoculation of young melon plants. Assessments of virus incidence were made using two different visual observation methods in concert with ELISA analyses of leaf disks samples collected biweekly. Infection rates were consistently lowest in plots treated with the butenolide insecticide flupyradifurone while dinotefuran was second in efficacy measures. Flupyradifurone also held whitefly densities to their lowest numbers relative to the other treatments. Two other insecticides, imidacloprid and cyantraniliprole, exacerbated virus incidence in multiple trials. Further investigation into the anomalous finding of increased virus incidence due to insecticide application is ongoing.

A Semipersistent Plant Virus Differentially Manipulates Feeding Behaviors of Different Sexes and Biotypes of Its Whitefly Vector.

It is known that plant viruses can change the performance of their vectors. However, there have been no reports on whether or how a semipersistent plant virus manipulates the feeding behaviors of its whitefly vectors. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emergent plant virus in many Asian countries and is transmitted specifically by B and Q biotypes of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. In the present study, we used electrical penetration graph (EPG) technique to investigate the effect of CCYV on the feeding behaviors of B. tabaci. The results showed that CCYV altered feeding behaviors of both biotypes and sexes of B. tabaci with different degrees. CCYV had stronger effects on feeding behaviors of Q biotype than those of B biotype, by increasing duration of phloem salivation and sap ingestion, and could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, with more phloem ingestion in Q biotype males and more non-phloem probing in B biotype males than their respective females. With regard to feeding behaviors related to virus transmission, these results indicated that, when carrying CCYV, B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females.

Mixed Infections of Four Viruses, the Incidence and Phylogenetic Relationships of Sweet Potato Chlorotic Fleck Virus (Betaflexiviridae) Isolates in Wild Species and Sweetpotatoes in Uganda and Evidence of Distinct Isolates in East Africa.

Viruses infecting wild flora may have a significant negative impact on nearby crops, and vice-versa. Only limited information is available on wild species able to host economically important viruses that infect sweetpotatoes (Ipomoea batatas). In this study, Sweet potato chlorotic fleck virus (SPCFV; Carlavirus, Betaflexiviridae) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus, Closteroviridae) were surveyed in wild plants of family Convolvulaceae (genera Astripomoea, Ipomoea, Hewittia and Lepistemon) in Uganda. Plants belonging to 26 wild species, including annuals, biannuals and perennials from four agro-ecological zones, were observed for virus-like symptoms in 2004 and 2007 and sampled for virus testing. SPCFV was detected in 84 (2.9%) of 2864 plants tested from 17 species. SPCSV was detected in 66 (5.4%) of the 1224 plants from 12 species sampled in 2007. Some SPCSV-infected plants were also infected with Sweet potato feathery mottle virus (SPFMV; Potyvirus, Potyviridae; 1.3%), Sweet potato mild mottle virus (SPMMV; Ipomovirus, Potyviridae; 0.5%) or both (0.4%), but none of these three viruses were detected in SPCFV-infected plants. Co-infection of SPFMV with SPMMV was detected in 1.2% of plants sampled. Virus-like symptoms were observed in 367 wild plants (12.8%), of which 42 plants (11.4%) were negative for the viruses tested. Almost all (92.4%) the 419 sweetpotato plants sampled from fields close to the tested wild plants displayed virus-like symptoms, and 87.1% were infected with one or more of the four viruses. Phylogenetic and evolutionary analyses of the 3'-proximal genomic region of SPCFV, including the silencing suppressor (NaBP)- and coat protein (CP)-coding regions implicated strong purifying selection on the CP and NaBP, and that the SPCFV strains from East Africa are distinguishable from those from other continents. However, the strains from wild species and sweetpotato were indistinguishable, suggesting reciprocal movement of SPCFV between wild and cultivated Convolvulaceae plants in the field.

Direct evidence for the semipersistent transmission of Cucurbit chlorotic yellows virus by a whitefly vector.

Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emerging plant virus, and is now spreading and causing severe economic losses to cucurbit crops in many Asian countries. CCYV is believed to be transmitted specifically by the sweetpotato whitefly, Bemisia tabaci, in a semipersistent manner. In the present study, we provide direct evidence for the semipersistent transmission of CCYV by Mediterranean (MED) cryptic species of B. tabaci complex. We investigated CCYV transmission characteristics, and immunofluorescently labeled and localized the virus retention site within the vector by laser confocal microscopy. Whiteflies required ≥1 h of acquisition access period (AAP) to successfully acquire CCYV, and the proportion of RT-PCR positive whitefly individuals reached to 100% at 48 h of AAP. CCYV virons could be retained within vectors as long as 12 d, but the proportion of RT-PCR positive whiteflies dropped to 55% by 3 d. Groups of thirty whiteflies given a 24 h of inoculation access period (IAP) to inoculate CCYV on cucumber plants showed a transmission efficiency rate of 72.73%. The retention site of CCYV virons was located in the foregut of virion-fed vectors. These results definitely indicated the semipersistent transmission mode of CCYV by B. tabaci MED.

Genetic diversity and silencing suppression activity of the p22 protein of Tomato chlorosis virus isolates from tomato and sweet pepper.

As for other bipartite criniviruses (genus Crinivirus, family Closteroviridae), the genome of Tomato chlorosis virus encodes an RNA silencing suppressor, the protein p22, in the 3'-proximal region of RNA1. This protein has been reported as having one of the longest lasting local suppressor activities when transiently expressed in Nicotiana benthamiana. Here, we examined the genetic diversity of the p22 gene in ToCV isolates from tomato and sweet pepper. The p22 gene sequences clearly grouped into two separated clades. However, functional analysis of both types of p22 proteins indicated no evident differences in suppressor activity. Our findings provide experimental evidence that the presence of a "strong" silencing suppressor is a conserved feature of ToCV isolates.

Nucleotide sequence and genome organization of a new proposed crinivirus, tetterwort vein chlorosis virus.

The genome of tetterwort vein chlorosis virus (TVCV) from South Korea has been completely sequenced. Its genomic organization resembles those of other criniviruses, with several new features, indicating that TVCV is a member of a new species in the genus Crinivirus, family Closteroviridae. RNA1 contains 8467 nucleotides, with at least four opening reading frames (ORFs). ORF1a encodes a protein with predicted papain-like protease, methyltransferase, and helicase activities. ORF1b encodes a putative RNA-dependent RNA polymerase that is apparently expressed through a +1 ribosomal frameshift. RNA2 contains 8113 nucleotides encoding at least nine proteins, similar to most crinivirus RNA2s. The 3' untranslated regions of the bipartite RNA genome share 82.1% nucleotide sequence identity.

Detection of Cucurbit chlorotic yellows virus from Bemisia tabaci captured on sticky traps using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and simple template preparation.

Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field.

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas†L.).

Three hundred and ninety-four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co-infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co-infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3-expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.