Direct Tissue Blot Immunoassay Research Articles

Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

Citrus yellow mottle-associated virus (CiYMaV) belonging to the subgenus Mandarivirus within the genus Potexvirus, was first identified in 2018 from Pakistan (CiYMaV-PK), where it is endemic in several regions. Here, three full-length cDNA clones (pCiYMaV-FL-1, pCiYMaV-FL-18, and pCiYMaV-FL-22) corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo (Citrus grandis) seedlings using the vacuum infiltration method. All the inoculated plants developed severe vein yellowing, leaf mottling, and dwarfing symptoms by 40 days post-infiltration (dpi). The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100% infection rates of pCiYMaV-FL at 60 dpi. Despite there being no observed difference among the three clones in the severity of symptom, pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins. Moreover, pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them. Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration. Taken together, the results indicate that Koch's postulates were fulfilled for CiYMaV in citrus plants. This is the first report of full-length infectious cDNA clones of CiYMaV, and thus, the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro

‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG). This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×104 to 1.4×108 cells μg–1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×108CLas cells μg–1 DNA were 30 and 40% at 30 and 40 DAG, respectively. Direct tissue blot immunoassay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in citrus plantlests in vitro. This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches.

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Development of Infectious cDNA Clones of Citrus Yellow Vein Clearing Virus Using a Novel and Rapid Strategy.

Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch's postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus-host interactions.

Citrus tristeza virus replication and movement in seedling trees of 71 rootstock genotypes

Citrus tristeza virus (CTV) replication and movement were studied in 1-year-old seedlings of 71 rootstock genotypesi, by inoculation with buds of an Imperial mandarin carrying multiple endemic strains of CTV including those causing seedling-yellows and quick-decline, but free of orange-stem-pitting strains. A virus-free Rough lemon bud was inserted 30-40 mm above the infected bud on each of the 965 nursery trees to study virus movement. A further subset of 226 trees of the same rootstock genotypes were budded with virus-free Imperial mandarin to serve as a control. Virus replication was detected (using direct tissue blot immunoassay) in most seedlings within six months of budding, with levels of infection indicating significant differences between nucellar selections, hybrid families, and within hybrid families. Genotypes lacking Poncirus in their pedigree were rapidly colonised by the virus, while those with Poncirus parentage were often either resistant or slow to replicate CTV. Large differences in the percentage of infected seedlings from Citrus x Poncirus hybrid families indicate that transmission of resistance is complex and not independent of the seed parent. CTV moved rapidly even in resistant genotypes with 100% of virus-free Rough lemon buds acquiring the virus within three months of budding. Tolerance to the diseases caused by CTV is an essential requirement of rootstocks used in Australia and this work has helped to describe initial virus replication in existing and potentially new commercial rootstocks.

Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

Improved method for assessing incidence of Citrus tristeza virus in large scale monitoring

Citrus tristeza virus (CTV) induces typical quick decline when citrus trees are grafted onto the sour orange. The virus is efficiently vectored by different aphid species . Large scale CTV monitoring and eradication requires a precise sampling and testing method for assessing virus incidence. The sampling procedure described by Hughes and Gottwald (1998) was adapted in the official monitoring for the mandatory control of CTV in the Apulia Region of Southern Italy. However, instead of the hierarchical sampling (HS) of grouped samples tested by Enzyme Linked Immunosorbent Assay (ELISA), a systematic sampling of individual trees tested by Direct Tissue Blot Immunoassay (DTBIA) was applied by the Mediterranean Agronomic Institute of Bari (MIAB-S). In this study, the actual CTV incidence assessed in the eight citrus commercial groves by DTBIA was used for evaluating the efficacy and precision of MAIB-S and HS methods. The simulation of different sampling scenarios using advanced spatial (automatic tree extraction) and statistical (Standard Error, Root Mean Square Error) analyses showed that in all groves the infection rates were always underestimated using the HS method, while most infection rates were overestimated and closer to the actual CTV incidence when the MAIB-S method was performed. For the MAIB-S method, no effect of high temperature in the detection of virus incidence occurred during July monitoring. Unlike MAIB-S, noticeable reduction in the precision of CTV incidence estimation was observed with HS.

Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees

A direct tissue blot immunoassay (DTBIA) technique has been compared with ELISA and PCR for detection of Xylella fastidiosa in olive trees from Apulia (southern Italy). Fresh cross-sections of young twigs and leaf petioles were printed onto nitrocellulose membranes and analyzed in the laboratory. Analyses of a first group of 61 samples gave similar efficiency for the three diagnostic techniques for detection the bacterium (24 positive and 36 negative samples), except for a single sample which was positive only with DTBIA and PCR. Similar results were obtained by separately analyzing suckers and twigs collected from different sectors of tree canopies of a second group of 20 olive trees (ten symptomatic and ten symptomless). In this second test the three diagnostic techniques confirmed the irregular distribution of the bacterium in the tree canopies and erratic detectability of the pathogen in the young suckers. It is therefore necessary to analyse composite samples per tree which should be prepared with twigs collected from different sides of the canopy. The efficiency comparable to ELISA and PCR, combined with the advantages of easier handling, speed and cost, make DTBIA a valid alternative to ELISA in large-scale surveys for occurrence of X. fastidiosa . Moreover, the printing of membranes directly in the field prevents infections spreading to Xylella -free areas, through movement of plant material with pathogen vectors for laboratory testing.

Comparison of different methods for Citrus tristeza virus detection in Satsuma mandarins

Citrus tristeza virus (CTV) infection in 13 Satsuma (Citrus unshiu (Mak.) Marc.) symptomless clones was investigated by different diagnostic methods, namely ELISA (Enzyme Linked Immunosorbent Assay), DTBIA (Direct Tissue Blot Immunoassay), and biological testing. The objective was to correlate the outcomes of the two serological tests mutually and with the severity of tristeza disease symptoms on indicator plants. ELISA and DTBIA were performed on the clones and on inoculated tissue samples of Mexican lime, while biological testing was performed on four citrus indicators. An additional goal of this study was to evaluate the efficiency of CTV DTBIA testing using different tissue types, including Satsuma immature ovaries and flower petioles. ELISA performed on spring shoots tissue of Mexican lime and DTBIA performed on mature ovaries of Satsuma field samples, showed high intensity of reactions and detection efficiency. Both tests were equally reliable and would make a good choice for fast CTV detection.

Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts

Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts.

Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

BackgroundCucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV.ResultsUsing the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA.ConclusionsThe established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.

Expression of recombinant Apple chlorotic leaf spot virus coat protein in heterologous system: production and use in immunodiagnosis

Expression condition for maximum recovery of recombinant Apple chlorotic leaf spot virus (ACLSV) coat protein was standardized. The in vitro expressed fusion protein with 6xHis tag (~43 Kd) was purified from inclusion bodies and used as an antigen for raising polyclonal antiserum in rabbit. This antiserum consistently detected ACLSV in pome and stone fruits as well as herbaceous host plants by direct double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and direct tissue blot immunoassay (DTBIA). The conditions for immuno-capture RT-PCR (IC-RT-PCR) were also standardized.

Occurrence of Viruses Affecting Potato Crops in Khartoum State-Sudan

The incidence of Alfalfa mosaic virus (AMV), Potato leafroll virus (PLRV), and Potato virus Y (PVY) in potato crops derived from various types of seed potatoes was assessed visually and confirmed by direct tissue blot immunoassay, over two winter growing seasons (1999/2000, 2000/2001) at three locations, Elnaiya, Elshehinab, and Shambat in Khartoum State, the main potato growing region in Sudan. Virus infection was most prevalent in 2000/2001. In general, crops grown directly from imported certified seed potatoes and from “improved seed”, produced in Sudan from imported basic seed, showed the lowest levels of PLRV and PVY compared with crops grown from Sudanese farm saved seed. For AMV, however, only crops grown directly from imported certified seed potatoes had low levels of AMV. Crop location also affected virus incidence, although this varied with year. For AMV, levels were similar at all locations in 1999/2000, but were greatest at Elnaiya in 2000/2001. For PVY, levels were greatest at Elnaiya in 1999/2000 and Shambat in 2000/2001. For PLRV, no symptoms were observed in 1999/2000 and virus levels were similar for all locations in 2000/2001. This study reports for the first time the occurrence of AMV in potatoes grown in Sudan.

RELIABILITY OF DIRECT TISSUE BLOT-ELISA AND DAS-ELISA IN THE DETECTION OF Citrus tristeza virus AND SAMPLING IN COMMERCIAL ORCHARDS IN TAMAULIPAS, MEXICO

The Citrus tristeza virus (CTV) causes a disease of regulatory interest for the Mexican citriculture. Timely and reliable sampling and diagnosis is essential for management strategies to check the spread of the brown citrus aphid, its main vector, in national territory. Because it is cheap and operationally feasible, direct tissue blot immunoassay-ELISA was compared with to DAS-ELISA, the official method for diagnosis, in order to establish an effective and efficient method for CTV detection. For the comparison, a total of 7421 trees of 11 commercial orchards of Tamaulipas, grouped by budding and infection age were tested. The direct tissue blot immunoassay technique was more sensitive and more capable of positive prognosis than DAS-ELISA in the diagnosis of CTV (P£0.028) in trees with recent and unknown infection. Buds diagnosed positive were heterogeneously distributed throughout the canopy, described by the beta binomial function (P£0.16-0.23). Based on this function and the reproducibility (93.2 %) of the results of double petiole printing suggested an optimal sample size of 10 petioles in single print. This method was 54.9 % more economical than DAS-ELISA and the diagnoses require only 17 % of the time invested for DAS-ELISA. However, due to the requirements of the current Mexican official norm (NOM-031-FITO-2000), this method is recommended as a fast technique to discriminate positive trees in field sampling to be later verified with the official method of diagnosis.

Detection of Citrus tristeza virus (CTV) from Satsuma Owari mandarins (Citris unshiu) by direct tissue blot immunoassay (DTBIA), DAS‐ELISA, and biological indexing

Three different citrus‐producing regions of Turkey—Edremit Gulf, Coastal Aegean, and eastern Black Sea—were surveyed in 2005 and 2006. A total of 119 samples were collected from Satsuma Owari mandarin (Citrus unshiu) trees grafted on Citrus tristeza virus (CTV)‐resistant Poncirus trifoliata rootstocks in commercial groves and home gardens. All samples were tested for the presence of CTV by direct tissue blot immunoassay (DTBIA) and double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) using young shoots. The samples that tested positive were indexed for biological properties. The results obtained from DTBIA tests showed that 20 of 119 (16.8%) tested trees were infected with CTV and 99 trees were virus free. All samples that tested positive in DTBIA were also positive in DAS‐ELISA. After biological indexing, none of these 20 isolates showed any symptoms on sour orange, grapefruit, and sweet orange plants, but all isolates induced vein‐clearing symptoms on Mexican lime (Citrus aurantifoli) in 9 months. Some isolates also caused leaf‐cupping and chlorosis in Mexican lime. However, 15 months after initial grafting, all isolates induced mild to moderate stem‐pitting on Mexican lime. In addition, young tissues, petioles, and leaf samples were collected periodically at monthly intervals for 1 year (2006) from a 25‐year‐old Satsuma Owari mandarin grafted on P. trifoliata in the Edremit Gulf. CTV was readily detected in tissue blots of young shoots and petioles from CTV‐infected plants during the autumn, winter, and spring seasons. Similarly, the highest ELISA values were obtained in April; the lowest values were noted in September. This study showed that DTBIA is a rapid, sensitive, and reliable procedure for detecting CTV under field conditions.

First Report from Syria of Citrus tristeza virus in Citrus spp.

During the spring of 2006, the main Syrian citrus-growing areas of Lattakia (Jableh, Aledyye, Eseelya, Siano, and Hresoon provinces) and Tartous (Almintar, Aljammase, Karto, Majdaloonelbahr, Yahmour, Amreet, Althawra, and Safita provinces) were surveyed to assess the presence of Citrus tristeza virus (CTV). Eight nurseries (approximately 130 plants per nursery), two budwood source fields (approximately 230 trees per field), and 19 groves (approximately 60 trees per grove) containing the main citrus varieties were visually inspected and sampled for serological assays. The hierarchical sampling method was carried out in each selected grove (2). Infected samples were collected from two nurseries, two budwood source fields, and six groves. Stems and leaf petioles from nursery trees and flower explants from the groves were collected and analyzed for CTV by direct tissue blot immunoassay (DTBIA) with the commercial kit from Plantprint (Valencia, Spain). Of 2,653 samples tested, 89 (4%) CTV-infected plants were detected. Five citrus varieties were found to be infected and Meyer lemon (Citrus limon 'Meyer') had the highest incidence at 16%. Numerous sweet orange varieties (Citrus sinensis L.) were found to be highly infected in the field, but only the Washington navel sweet orange was found to be infected in the nurseries. No clear CTV symptoms were observed during the survey. Samples that were positive for CTV by DTBIA were also positive by biological indexing on Mexican lime (C. aurantifolia) and immunocapture-reverse transcription-PCR as described by Nolasco et al. (3). Coat protein gene sequences obtained from five selected clones of a Syrian CTV isolate (GenBank Accession No. EU626555) showed more than 99 and 98% nucleotide sequence identity to a Jordanian CTV isolate (GenBank Accession No. AY550252) and the VT isolate (GenBank Accession No. U56902), respectively. Almost all infected samples induced moderate vein clearing symptoms when grafted to Mexican lime. Symptoms of vein clearing, leaf cupping, stunting, and stem pitting on Mexican lime were induced by graft transmission of CTV from one Valencia sample from the Tartous area. The viral inoculum is widely and randomly distributed in commercial groves, especially in the southern Tartous area and in some nurseries. To our knowledge, this is the first report of CTV in Syria. However, CTV was reported from the neighboring citrus-growing countries of Lebanon, Turkey, and Jordan (1), and the severe seedling yellows strain is present in this area, which poses a potential threat to Syrian citriculture.

Prereaction of Citrus tristeza virus (CTV) Specific Antibodies and Labeled Secondary Antibodies Increases Speed of Direct Tissue Blot Immunoassay for CTV.

An improved direct tissue blot immunoassay (DTBIA) procedure for detection of Citrus tristeza virus (CTV) within 1 h is described. Prints of fresh young stems of citrus plants that were infected or not infected with CTV were made by gently and evenly pressing the fresh-cut surface of the stems onto a nitrocellulose membrane. The tissue blots were air-dried for 5 min, incubated with prereaction solutions of CTV-specific antibodies and labeled secondary antibodies, goat anti-mouse Ig (H+L)-alkaline phosphatase conjugate or goat anti-rabbit IgG alkaline phos-phatase conjugate, for up to 20 min, rinsed with PBST buffer for 5 min, and immersed into an NBT-BCIP substrate solution for 15 to 20 min. Then the blots were rinsed in water for a few seconds to stop the reactions, and the results were observed and recorded under a light microscope. All samples from greenhouse plants that were infected with CTV decline inducing isolate T-36 were positive to CTV-specific polyclonal antibody 1212 (PCA 1212) and monoclonal antibodies 17G11 (MAb 17G11) and MCA13 (MAb MCA13), whereas samples from greenhouse plants infected with non-decline-inducing isolate T-30 were positive to PCA 1212 and MAb 17G11, but not to MAb MCA13. The noninfected greenhouse plants were negative to all of the antibodies. The improved DTBIA was at least as reliable as other immunological procedures and almost as reliable as polymerase chain reaction for detecting CTV in field trees. The improved DTBIA enables the detection of CTV within 1 h by having a prereaction of CTV-specific antibodies and labeled secondary antibodies in solutions before they are applied to the tissue blots. This DTBIA procedure may be useful in detecting other plant viruses and other pathogens such as bacteria and fungi.

Preliminary Studies on CPG/ Hinf I RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus (CTV) causes economically important losses to the citrus industry worldwide. Mild strain cross protection (MSCP) against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces (Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi) were tested by direct tissue blot immuno-assay (DTBIA), and 158 of them were tested positively, which therefore were subjected to coat protein gene (CPG)/ Hinf I restriction fragment length polymorphism (RFLP) analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/ Hinf I RFLP groups III and I are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/ Hinf I RFLP groups III and I caused severe stem-pittings in sweet oranges in China.

Occurrence and distribution of Citrus tristeza virus (CTV) in the Jordan Valley

In a survey conducted in 2002 and 2003, Citrus tristeza virus (CTV) was detected in the Jordan Valley. The direct tissue blot immunoassay (DTBIA) indicated that 12.7 and 15.2% of samples tested in the central and northern Jordan Valley respectively were infected with CTV. Similar results showed that all citrus species grown in the Jordan Valley were susceptible to CTV. DAS-ELISA analysis of samples from a citrus orchard in the Dir Alla area with severe CTV symptoms indicated that 49% of samples were infected with CTV. Using a CTV specific primer pair (CTV1/CTV10), the coat protein gene of the virus was successfully amplified from leaf extracts obtained from CTVinfected trees by IC-RT-PCR. After cloning and sequencing the coat protein gene, the sequence of the amplified product was deposited in the GenBank.