Acquisition Access Period Research Articles

Competence of Frankliniella occidentalis and Frankliniella intonsa strains as vectors for Chrysanthemum stem necrosis virus

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.

High Viral Load in the Planthopper Vector Delphacodes kuscheli (Hemiptera: Delphacidae) is Associated With Successful Transmission of Mal de Río Cuarto Virus

Abstract Delphacodes kuscheli Fennah (Hemiptera: Delphacidae) is the main natural vector of Mal de Rio Cuarto virus (family Reoviridae, genus Fijivirus, MRCV), which infects different gramineae and causes the most important maize (Zea mays L.) disease in Argentina. MRCV—vector interactions usually are studied using different winter cereals as hosts. Under experimental conditions, <50% of D. kuscheli planthoppers fed on a MRCV-infected plant can transmit the virus to wheat (Triticum aestivum L. ). This fact is influenced by insect development stage at acquisition and the latency period. This work describes the relation between transmission efficiency and MRCV accumulation in its planthopper vector. First- and third-instar D. kuscheli nymphs were allowed to feed on MRCV-infected plants, and 9 or 17 d after the acquisition access period (AAP), viral load of transmitting and nontransmitting planthoppers was quantified by quantitative polymerase chain reaction. The transmitting planthoppers showed significantly higher viral titers than nontransmitting ones, suggesting that successful transmission is positively associated to viral accumulation in the insect. However, planthoppers of the third-instars group did not transmit the virus 9 d after AAP, even when 46% had similar titers to the transmitting insects of the other treatments. These results indicate that additional factors influence MRCV transmission efficiency when acquisition occurs in older planthoppers. This is the first precise quantitative analysis of MRCV in its main vector species and will definitely contribute to better understand planthopper—Fijivirus interactions and its epidemiological implications.

Stylet penetration behaviour of Pseudococcus longispinus in relation to acquisition of grapevine leafroll virus 3

Grapevine leafroll–associated virus-3 (GLRaV-3) is transmitted from infected grapevines to healthy vines by mealybugs. The stylet penetration behaviour of Pseudococcus longispinus (Hemiptera: Pseudococcidae) was electronically monitored on leaves of grape plants to quantify the time taken to access the phloem and the duration of phloem ingestion. More of the first instar nymphs than of the adults showed sustained (>10 min) phloem ingestion; however, nymphs were affected adversely by longer monitoring periods. The probing and phloem ingestion periods of adults on GLRaV-3-infected Red Globe and uninfected Pinot noir were not significantly different from each other. Most adults showed the first phloem ingestion and the first sustained phloem ingestion after 12 h and after 24 h of their first probe, respectively. Free (unwired) and electrical penetration graph (EPG)-monitored (wired) P. longispinus adults were tested for acquisition of virus after different acquisition access periods (AAP). Acquired GLRaV-3 RNA in extracts of whole insects was detected by reverse transcription and polymerase chain reaction amplification. For the unwired adult mealybugs, 30 % that had an AAP of 48 h and 90 % that had an AAP of 72 or 96 h were positive for GLRaV-3. Five out of 24 (21 %) of the wired mealybugs allowed at least 24 h APP (1.8–44.4 h of phloem ingestion) were positive for GLRaV-3. None of the adult mealybugs acquired virus during the first 24 h of AAP. We conclude that GLRaV-3 acquisition by P. longispinus generally occurs within 24–72 h AAP with as little as 1.8 h of phloem ingestion.

Transmission Efficiency of ‘Candidatus Liberibacter solanacearum’ and Potato Zebra Chip Disease Progress in Relation to Pathogen Titer, Vector Numbers, and Feeding Sites

ABSTRACT With diseases caused by vector-borne plant pathogens, acquisition and inoculation are two primary stages of the transmission, which can determine vector efficiency in spreading the pathogen. The present study was initiated to quantify acquisition and inoculation successes of 'Candidatus Liberibacter solanacearum', the etiological agent of zebra chip disease of potato, by its psyllid vector, Bactericera cockerelli (Hemiptera: Triozidae). Acquisition success was evaluated in relation to feeding site on the host plant as well as the acquisition access period. Inoculation success was evaluated in relation to vector number (1 and 4) on the plants. Acquisition success was influenced by the feeding site on the plant. The highest acquisition success occurred when insects had access to the whole plant. The results of the inoculation study indicated that the rate of successfully inoculated plants increased with the vector number. Plants inoculated with multiple psyllids had higher bacterial titer at the point of inoculation. Although disease incubation period was significantly shorter in plants inoculated with multiple psyllids, this effect was heterogeneous across experimental blocks, and was independent of pathogen quantity detected in the leaflets 3 days postinoculation. Disease progress was not affected by bacterial quantity injected or psyllid numbers.

Whitefly-mediated transmission of cotton leaf curl Multan betasatellite: evidence for betasatellite encapsidation in coat protein of helper begomoviruses

Cotton leaf curl Multan betasatellite (CLCuMB) is responsible for symptom expression of a devastating disease of cotton in the Indian subcontinent. CLCuMB depends on helper virus replication-associated protein for its replication and on viral coat protein (CP) for its encapsidation. However, no direct evidence of encapsidation of CLCuMB in viral CP has been available. In the present study, non-viruliferous whiteflies were placed on tomato plants that had been agroinoculated with infectious clones of an Iranian isolate of tomato yellow leaf curl virus (TYLCV-[Ab]) and CLCuMB for an acquisition access period of 72 h and then transferred to healthy tomato seedlings at the 3- to 4-leaf stage. Typical symptoms of TYLCV-[Ab] appeared on inoculated seedlings 30-45 days post-inoculation. The presence of TYLCV-[Ab] and CLCuMB DNAs in symptomatic test plants and viruliferous whiteflies was confirmed by PCR analysis using specific primers and DIG Southern blotting. Furthermore, the possibility of CLCuMB DNA encapsidation in TYLCV-[Ab] CP within infected plants was examined by immunocapture PCR. The results showed that CLCuMB DNA was encapsidated in TYLCV-[Ab] CP. Whitefly-mediated transmission of CLCuMB in the presence of helper virus is additional evidence for encapsidation of CLCuMB by TYLCV-[Ab] CP.

Transmission characteristics of Southern rice black-streaked dwarf virus by rice planthoppers

Southern rice black-streaked dwarf virus (SRBSDV) is a recently proposed distinct species in the genus Fijivirus, family Reoviridae. During the past decade, SRBSDV has spread throughout southern China and northern Vietnam, and has become one of the greatest threats to rice production in these regions. We evaluated three common planthopper species affecting rice: white-backed planthopper (WBPH, Sogatella furcifera), brown planthopper (BPH, Nilaparvata lugens) and small brown planthopper (SBPH, Laodelphax striatellus) to determine their virus transmission abilities. It was confirmed that WBPH was an efficient persistent-transmitting vector for SRBSDV. Neither BPH nor SBPH were viral vectors, although a small proportion (3.7%) of tested SBPH acquired the virus from diseased rice. We characterized the virus transmission properties of WBPH. 83% of the tested insects fed on virus-infected rice plants became viruliferous. The minimum virus acquisition and inoculation access periods were 5 and 30 min, respectively, for both WBPH nymphs and adults. The circulative transmission periods of the virus in WBPH ranged from 6 to 14 days, and most viruliferous individuals transmitted the virus in intermittent periods ranging from 2 to 6 days. A single individual of WBPH could infect 8–25 rice plants with the virus in a 5-day period. WBPH could transmit SRBSDV from rice to maize seedlings, but it was barely able to acquire the virus from infected maize. These results improve our understanding of the epidemiology of SRBSDV, and will be useful for development of disease control strategies.

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response toTomato Spotted Wilt VirusInfection

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Semipersistent Whitefly Transmission of Squash vein yellowing virus, Causal Agent of Viral Watermelon Vine Decline.

Squash vein yellowing virus (SqVYV), a recently described Ipomovirus sp. in the family Potyviridae, is the cause of viral watermelon vine decline, a devastating disease in Florida. SqVYV is known to be transmitted by the whitefly, Bemisia tabaci (Gennadius) B strain, but details of the transmission process have not previously been investigated. We completed a series of experiments to determine efficiency of transmission, effects of different acquisition and inoculation access periods, the length of time that whiteflies retained transmissible virus, and the minimum time needed to complete a cycle of acquisition and inoculation. Efficiency was low, with at least 30 whiteflies per plant needed for consistent transmission. Acquisition leading to later transmission peaked at 4 h, and inoculation access periods longer than 4 to 8 h led to no increase in infection rates. Whiteflies retained virus only a short time, with no transmission by 24 h after removal from infected plants. A minimum of 3 h was needed to complete a cycle of transmission under laboratory conditions. These results demonstrate semipersistent transmission of SqVYV and will help refine models of the epidemiology of this virus and the disease it causes.

First Report of Tomato chlorosis virus in Potato in Brazil.

Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system.

Effects of Gender, Origin, and Age on Transmission of Xylella fastidiosa to Grapevines by Homalodisca vitripennis (Hemiptera: Cicadellidae)

Abstract Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) is an invasive vector of Xylella fastidiosa, the causal agent of Pierce's disease of grapevines (Vitis vinifera L.). Transmission efficiency of X. fastidiosa to grapevines by H. vitripennis originating from two geographically separated populations in California (Riverside and Bakersfield) based on gender and age was evaluated. To evaluate transmission efficiencies among groups, insects were given a 96-h acquisition access period on infected grapevines and caged in groups of five on healthy grapevines for a 72-h inoculation access period. At the conclusion of tests, polymerase chain reaction determined that a mean ± SD of 0.98 ± 1.02 X. fastidiosa-positive H. vitripennis were present on each grapevine and that 29% of test plants were infected. Acquisition and retention of X. fastidiosa was not affected by vector origin, gender, or age. The probability that a grapevine was infected 12 wk after the inoculation access period increased with the number of X. fastidiosa-positive H. vitripennis per plant. Gender had no effect on inoculation success and main effects of insect origin and age on inoculation success were minor. Collectively, the result indicated that H. vitripennis origin, gender, and age did not affect acquisition and retention of X. fastidiosa and had only minor effects on inoculation success under laboratory conditions. Accordingly, rates of pathogen spread in the field are more likely to be affected by local biotic and abiotic factors that differentially affect H. vitripennis abundance and movement based on origin, gender, and age than inherent differences in transmission efficiencies among these groups.

Molecular Studies on the Transmission of Indian Cassava Mosaic Virus (ICMV) and Sri Lankan Cassava Mosaic Virus (SLCMV) in Cassava by Bemisia tabaci and Cloning of ICMV and SLCMV Replicase Gene from Cassava.

Cassava mosaic disease, caused by cassava mosaic geminiviruses are transmitted by Bemisia tabaci. The B. tabaci adults from colonies reared on virus free cassava plant produced from apical meristem culture was studied to determine their ability to transmit Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) from cassava to cassava. Virus free plants were confirmed by polymerase chain reaction (PCR) using geminivirus degenerate primers. The virus acquisition access period (AAP) of 48h on virus infected cassava leaves and 48h virus inoculation access periods on virus free healthy leaves were investigated. Both ICMV and SLCMV were absolutely transmitted by whiteflies reared on cassava. Virus specific primers were designed in the replicase region and used to detect virus in B. tabaci after different AAP. The PCR amplified replicase genes from virus transmitted cassava leaves were cloned the plasmid DNA was isolated from a recombinant colony of E. coli DH5α after their confirmation by colony PCR and sequenced them. The nucleotide sequences obtained from automated DNA sequencing were confirmed as ICMV and SLCMV replicase gene after homology searching by BLAST and found to be a new isolates. The nucleotide sequences of new isolates were submitted in GenBank (accession number JN652126 and JN595785).

Bemisia tabaci Phylogenetic Groups in India and the Relative Transmission Efficacy of Tomato leaf curl Bangalore virus by an Indigenous and an Exotic Population

Bemisia tabaci adults from various host-plant species were collected from 31 regions across India. 266 B. tabaci samples were first screened by RAPD-PCR to examine molecular variability and to select individuals with different fingerprints. Host-plant and region of collection were then used to select 25 individuals for PCR amplification and sequencing of their partial mitochondrial cytochrome oxidase subunit one (mtCOI) genes. Pairwise comparisons with mtCOI consensus sequences showed that the majority of these samples had <3.5% sequence divergence from groups currently termed Asia I, Asia II-5, Asia II-7, and Asia II-8. The biotype-B B. tabaci from India clustered into the Middle East-Asia Minor 1 group. A new group of B. tabaci from Coimbatore, collected from pumpkin, was related most closely to the Asia I group (6.2% sequence divergence from the consensus Asia I sequence). To increase our understanding of the epidemiology of tomato leaf curl disease (ToLCD) and the key B. tabaci genetic groups involved in virus spread, the indigenous Asia I and the exotic biotype-B population from South India were used to carry out transmission experiments using Tomato leaf curl Bangalore virus (ToLCBV). The acquisition access periods (AAP), inoculation access periods (IAP), latent periods (LP), and ToLCBV transmission efficiencies of the two populations were compared and the biotype-B had the more efficient transmission characteristics. These results are discussed in relation to recent changes in the epidemiology of tomato leaf curl disease in South India.

First Report of Turnip mosaic virus in Tomatillo (Physalis philadelphica) in California.

Tomatillo is an important vegetable in Mexican cuisine. It is of Mesoamerica origin and now is grown widely in the Western Hemisphere. In 2011, 2% of commercially grown tomatillo plants in San Benito County, California exhibited severe stunting with foliage showing mosaic symptoms and leaf distortion. The fruits on infected plants were mottled and unmarketable. Flexuous filamentous-shaped virus particles of 800 to 850 nm long and 11 to 12 nm wide were observed from sap of the symptomatic plants with a transmission electron microscope. Sap from the diseased tomatillo plants reacted positively in an immunostrip assay for potyvirus (Agdia Inc., Elkhart, IN), indicating a potyvirus was associated with the disease. The causal agent was mechanically transmitted from the diseased field plants to six virus-free greenhouse tomatillo plants and all inoculated plants induced identical symptoms. The causal agent was also transmitted to Chenopodium quinoa and C. murale (chlorotic local lesions) and Nicotiana clevelandii, N. tabacum, and Physalis wrightii (systemic symptoms). The disease was also transmitted to tomatillo plants by the green peach aphid (Myzus persicae) in a nonpersistent manner (1-min acquisition access period and 1-min transmission access period with no latent period). To further identify the causal agent, total nucleic acids were extracted by a cetyltrimethylammoniumbromide (CTAB) method (2) and tested by reverse transcription-PCR using potyvirus degenerate primers CIFor and CIRev (1). An amplicon of approximately 700 bp from the diseased tomatillo was cloned and sequenced. Analysis of the 631-bp partial CI sequence (GenBank Accession No. JN601884) showed that the virus had 93.6% nucleotide identity and 100% amino acid identity with cognate regions of Turnip mosaic virus (TuMV) (GenBank Accession No. D10927). Our results indicated that the disease was caused by TuMV. To our knowledge, this is the first report of TuMV in tomatillo. Since TuMV has a wide host range and is readily transmitted by green peach aphids, TuMV could be a new threat to tomatillo production in California.

Virus–vector Relationships, Host Range, Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur, India

AbstractAn epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli‐growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.

Detection and Variability of Aster Yellows Phytoplasma Titer in Its Insect Vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae)

The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper, Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual aster leafhoppers, we developed a quantitative real-time polymerase chain reaction assay to measure AYp concentration in insect DNA extracts. Absolute quantification of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (tuf), from whole insect DNA extractions, to the amplification of a dilution series containing known quantities of the tuf gene sequence cloned into a plasmid. The capabilities and limitations of this method were assessed by conducting time course experiments that varied the incubation time of AYp in the aster leafhopper from 0 to 9 d after a 48 h acquisition access period on an AYp-infected plant. Average AYp titer was measured in 107 aster leafhoppers and, expressed as Log10 (copies/insect), ranged from 3.53 (+/- 0.07) to 6.26 (+/- 0.11) occurring at one and 7 d after the acquisition access period. AYp titers per insect and relative to an aster leafhopper chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. High quantification cycle values obtained for aster leafhoppers not exposed to an AYp-infected plant were interpreted as background and used to define a limit of detection for the quantitative real-time polymerase chain reaction assay. This method will improve our ability to study biological factors governing AYp replication in the aster leafhopper and determine if AYp titer is associated with frequency of transmission.

Tomato yellow leaf curl virus (TYLCV), identification, virus vector relationship, strains characterization and a suggestion for its control with plant extracts in Iraq

Tomato yellow leaf curl begomovirus (TYLCV) was identified on the basis of symptoms on test plants, transmission by whiteflies Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) biotype B and its incubation period in the insect. Symptomatic tomato plants showing leaf curling, leaf blade reduction, distortion and general stunting, indicative of TYLCV in greenhouses, were transplanted into pots (20 × 20 cm) containing soil mix and were maintained in growth room for identification. The virus was found to be easily acquired and transmitted by B. tabacibiotype B when given an acquisition access period on tomato infected plants for 30 min with incubation period of 24 h. Leaf curl and distortion associated with severe stunting and yellowing were developed on inoculated Datura stramonium, Nicotiana tabacum cv. Samsun NN, Nicotiana glutinosa and Phaseolus vulgaris var. Battle and were found as susceptible to the virus without visible symptoms. The virus caused vein clearing on inoculated Solanum nigrum leaves. No infections were observed on Gomphrena globosa, N. tabacum cv. White burly, Physalis floridana, Solanum melongena, Cucumis sativus, Chenopodium amaranlicolor and Chenopodium murale inoculated by the virus. Two strains for TYLCV were characterized by symptoms on tomato and immunu-double diffusion test. A spur was formed between the polyclonal antibodies of TYLCV isolate and the extracts from infected tomato plants showing different symptoms. Extracts from Thuja, Tamarix and Henna plants exhibited inhibitory effects on TYLCV multiplication in tomato treated plants with protection periods of 10 to 12 days. Key words: Bemisia tabaci, control, plant extracts, strains, Tomato yellow leaf curl begomovirus.

First Report of Cucurbit chlorotic yellows virus Infecting Muskmelon and Cucumber in Sudan.

In summer 2009, a survey for virus diseases in cucurbits was conducted in open fields and plastichouses in Khartoum State, the most important growing area for cucurbits in Sudan. Chlorosis and yellowing symptoms on middle and lower leaves were observed on many muskmelon (Cucumis melo L.) plants grown in open fields in the Assilat agricultural scheme and on approximately 80% of the cucumber (Cucumis sativus L.) plants grown in plastichouses in Khartoum North. Large populations of whiteflies (Bemisia tabaci L.) were present in both locations. Leaf symptoms that were observed were similar to those caused by Cucurbit chlorotic yellows virus (CCYV), a recently described new Crinivirus species infecting cucurbits in Japan (4), indicating presence of this virus previously only reported from Japan, Taiwan (2), and China (1). Samples from seven symptomatic muskmelon leaves were collected from individual plants grown in different open fields in Assilat and from a symptomatic cucumber plant grown in a plastichouse. Total RNA was extracted from these samples with the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) to amplify putative CCYV sequences with primers (Crini-s2 5'-CATTCCTACCTGTTTAGCCA and Crini-as2 5'-TGCACTTATAATCTGCTGGTAC) designed from CCYV sequences available at GenBank. A 353-bp DNA fragment of the HSP70 gene was amplified by reverse transcription (RT)-PCR for all samples. Further analysis by direct sequencing of two PCR products showed 99 to 100% nt sequence identity to Asian CCYV isolates. Amplification of the coat protein sequence with the primer pair (CCYV-CPs 5'-ATGGAGAAGACTGACAATAAACAA and CCYV-CPas 5'-TTTACTACAACCTCCCGGTG) followed by cloning and sequencing yielded a 760-bp fragment having 99% nucleotide sequence identity to all Asian isolates. For confirmation, dsRNA preparations of symptomatic muskmelon tissue (collected in June 2010) were made, showing dsRNA patterns typical for criniviruses after separation on agarose gels. This dsRNA was used as template for random RT-PCR followed by sequencing of the cloned PCR products (3). Comparison with sequences available at GenBank revealed that cDNA sequences from dsRNA also were 99 to 100% identical to the CCYV genome sequence (AB523788.1). Whitefly transmission of the virus was confirmed by giving a population of B. tabaci an acquisition access period of 24 h and a further 24 h on muskmelon and cucumber seedlings. Symptoms were observed after 5 to 7 days, and the presence of CCYV was confirmed by RT-PCR. In conclusion, symptoms, RT-PCR, and dsRNA sequencing results confirm the presence and establishment of CCYV in cucurbit crops in Sudan. It is remarkable that the sequences obtained from the Sudanese samples show only negligible sequence differences from Asian isolates. Because of the large whitefly vector populations, the spread of CCYV to neighboring countries in Africa and potentially southern Europe, or wherever cucurbits are grown, can be expected. To our knowledge, this is the first report of CCYV in Sudan and outside Eastern Asia. The sequences obtained in this study have been submitted to GenBank under Accession Nos. JF807053 to JF807055.

Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conserved fragments of the polymerase region of each virus and were effective for the detection of different isolates tested in this study. The RBDV assay amplified a 94bp amplicon and was able to detect as few as 30 viral copies. Whereas the RLMV assay amplified a 180bp amplicon and detected as few as 300 viral copies from plant and aphid RNA extracts. Both assays were significantly more sensitive than their corresponding conventional RT-PCR methods. The sensitivity of the RLMV assay was also tested on single aphids after a fixed acquisition access period (AAP). In addition, the assays revealed a novel synergistic interaction between the two viruses, where the concentration of RBDV was enhanced ∼400-fold when it occurred in combination with RLMV compared to its concentration in single infections. The significance of this finding and the importance of the development of real-time RT-PCR assays for the detection of RBDV and RLMV are discussed.

Assessing aphids potato virus Y-transmission efficiency: A new approach

In order to develop an alternative method to optimize the relative efficiency factor (REF) assessment, the efficiency of transmission of Potato virus Y (PVY) by seven aphid species was examined. In vitro micropropagated potato plantlets were used to experiment on phenotypically and genetically homogeneous material. Species-specific acquisition access period (AAP) on a PVY-infected plantlet was assessed for each aphid species using electrical penetration graph (EPG) technique.Aphid probing behaviour determined by EPG showed that Macrosiphum euphorbiae and Myzus persicae exhibited the shortest AAPs (15 and 11min, respectively) whereas Rhopalosiphum padi, Sitobion avenae, Brevicoryne brassicae and Acyrthosiphon pisum exhibited the longest ones (more than 30min). The transmission rate obtained for M. persicae (83.3%) was higher than the ones reported in the literature. REFs assessment showed that A. pisum and B. brassicae were poor efficient vectors while M. euphorbiae and S. avenae seemed to be efficient ones even though their respective REF were significantly lower than that of M. persicae. The species R. padi and A. fabae did not transmit PVY.The hypothesis assessed for M. euphorbiae and S. avenae and consisting in the compensation of a weak PVY-transmission efficiency by a higher number of vectors, was not supported. The use of this new method for REF evaluation and the need to consider aphid behaviour for such an assessment was discussed.

Acquisition and Transmissibility of U.S. Soybean dwarf virus Isolates by the Soybean Aphid, Aphis glycines.

Soybean dwarf virus (SbDV) exists as several distinct strains based on symptomatology, vector specificity, and host range. Originally characterized Japanese isolates of SbDV were specifically transmitted by Aulacorthum solani. More recently, additional Japanese isolates and endemic U.S. isolates have been shown to be transmitted by several different aphid species. The soybean aphid, Aphis glycines, the only aphid that colonizes soybean, has been shown to be a very inefficient vector of some SbDV isolates from Japan and the United States. Transmission experiments have shown that the soybean aphid can transmit certain isolates of SbDV from soybean to soybean and clover species and from clover to clover and soybean with long acquisition and inoculation access periods. Although transmission of SbDV by the soybean aphid is very inefficient, the large soybean aphid populations that develop on soybean may have epidemiological potential to produce serious SbDV-induced yield losses.