Citrus Tristeza Virus Incidence Research Articles

Incidence of Citrus tristeza virus in Zaria and Giwa Local Government Areas of Kaduna State, Nigeria

Citrus tristeza disease is among the most devastating diseases of citrus worldwide. To ascertain the occurrence of the causal agent, Citrus tristeza virus (CTV) in Zaria and Giwa Local Government Areas of Kaduna State, samples were collected from four citrus orchards in September during the 2022 wet season. A total of sixty samples (n=60) were collected randomly comprising of both symptomatic and asymptomatic leaves, fifteen (n=15) from each field. The commonest symptoms observed included, yellowing of leaves, cupping, and leave vein clearing. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was employed in the detection of the virus from the leaves collected. The result shows the presence of the virus in the Local Government Areas (LGAs) with an incidence of 6.67% each. This study is the first to report the presence of CTV in Zaria and Giwa LGAs of Kaduna State. Thus, indicating the spread of the viral disease in the country-despite its low incidence- after it was first reported from the Southern part of Nigeria. This calls for an intensive survey to delineate the extent of the viral incidence in the country and embark on enlightenment campaigns for citrus farmers.

First comprehensive sanitary report of citrus-infecting viruses and viroids in Uruguay

Citrus is the highest-value fruit crop in terms of international trade. However, citrus species are susceptible to several diseases caused by different pathogens which directly cause a decrease in production leading to economic losses. In the last half-century, the citrus industry in Uruguay has had a strong socio-economic impact and is also constantly evolving to stay competitive in world markets, by introduction of new varieties and improvement of production practices to obtain high yielding orchards. Nevertheless, despite the existence since 2014 of the Uruguayan National Citrus Sanitary and Certification Program, scarce information is available regarding the virus and viroid status of commercial citrus in Uruguay. The incidence of citrus tristeza virus (CTV), citrus psorosis virus (CPsV), satsuma dwarf virus (SDV), citrus exocortis viroid (CEVd), hop stunt viroid (HSVd), citrus dwarfing viroid (CDVd), citrus bark cracking viroid (CBCVd) and citrus bent leaf viroid (CBLVd) was investigated in this study, as well as CTV genotypes prevalent in the country. Molecular diagnostic assays were used to test 1175 samples including Valencia and Navel sweet oranges, Mandarin hybrids, Clementines and lemons, which were randomly collected from seven citrus-producing provinces. Only 6% of the samples were negative for the pathogens screened, while 93% of them were CTV positive. SDV, CBLVd and CBCVd were not detected. Co-infections were frequently detected, finding plants with up to four simultaneous pathogens, including CTV in all co-infected plants. This is the first comprehensive survey of several citrus-infecting viruses and viroids in Uruguay, as well as a determination of the CTV genotypes prevalent in the country.

Prevalence of mild citrus tristeza virus isolates of the T30 genotype in Cuban commercial citrus fields after the dissemination of huanglongbing

Huanglongbing (HLB) disease detection in Cuba in 2006 led to a turn in citrus management in the country, including the production of certified budwood in protected nurseries, eradication of symptomatic plants and old orchards, and chemical control of the vector Diaphorina citri. This dramatic shift in citrus farming could have affected the incidence and composition of citrus tristeza virus (CTV) populations. This work aimed to determine CTV incidence and characterize viral isolates from the main Cuban citrus-producing areas. A survey throughout the country was carried out which resulted in the collection of 27 CTV isolates. Results showed a decrease of CTV incidence in western and central areas of the country, but an increase in the East, compared to previous reports. Bioindexing showed that most of the isolates induced none or mild symptoms. Serological characterization using seven different CTV specific monoclonal antibodies (MAb) showed heterogeneity among the 27 isolates. Six out of twenty-seven were MCA13-reactive, a MAb associated to the presence of severe CTV isolates. Nevertheless, sequence comparison of the p20, p23 and p25 genes evidenced a high nucleotide sequence identity among isolates, and according to the phylogenetic analyses, all the sequences belonged to the CTV T30 genotype. Thus, our results showed a decrease in the CTV incidence in the surveyed areas and the broad dissemination of mild CTV isolates of the T30 genotype, which could be partially explained by the shift in the management programs implemented for CTV and HLB control in Cuba.

Spread of Citrus Tristeza Virus in Citrus Orchards in Central California.

In California, citrus tristeza virus (CTV) is regulated by a State Interior Quarantine. In CTV abatement districts in central California, trees with CTV that react to MCA13 (MCA13-positive [MCA13+]), a strain-discriminating monoclonal antibody, are rogued to prevent virus spread. The Tulare County Pest Control District, however, does not participate in this abatement program except for a 1.6-km2 zone around the Lindcove Research and Extension Center, Exeter, CA. To quantify CTV spread under these two disparate management programs, CTV surveys were conducted in abatement plots with mandatory aphid control and nonabatement plots. Abatement plot surveys used hierarchical sampling of 25% of trees with samples pooled from four adjacent trees. Detection of MCA13+ CTV in a sample prompted resampling and testing of individual trees. From 2008 to 2018, incidence of CTV increased by an average of 3.9%, with only two MCA13+ samples detected. In contrast, in nonabatement plots, incidence of CTV increased by an average of 4.6% between 2015 and 2018. Increase in MCA13-negative (MCA-) isolates was 11 times greater than that of MCA13+ isolates, with the number of MCA13+ trees increasing by 19 trees between 2015 and 2018. MCA13- isolates were more randomly distributed, suggesting primary spread, whereas MCA13+ CTV isolates were more aggregated, suggesting some secondary spread. These results suggest that spread of MCA13+ isolates was limited by a combination of tree removal and aphid vector suppression. MCA13+ samples were VT isolates with some mixtures with T30 isolates. Despite the presence of VT isolates, all CTV-infected trees were asymptomatic.

Molecular detection and coat protein gene based characterization of Citrus tristeza virus prevalent in Sikkim state of India

Citrus tristeza virus (CTV), an important graft transmissible pathogen infecting citrus (Citrus spp.) is prevalent in different citrus growing areas of the country. Sikkim is one of important citrus producing states in the Northeast region of India where Khasi mandarin (C. reticulata) is commercially cultivated. Apart from this, there is limited plantation of Assam lemon (C. limon), Rangpur lime (C. limonia) and Pomelo (C. grandis) in this region. We performed molecular characterization based on coat protein sequence analysis of CTV isolates from Sikkim state of India. Thirty six field samples suspected with Tristeza disease were tested by reverse transcription-polymerase chain reaction (RT-PCR) using CP specific primers (CN150/CN151). Out of 36, 11 samples were found CTV positive. Eight of the 36 samples showing prominent field symptoms were bio-indexed on Mexican lime (C. aurantifolia) indicator plant for CTV. Five of them showed CTV specific vein clearing and vein flecking symptoms. CTV incidence of 30.55% was recorded in the Sikkim based on combined results of ELISA and PCR. In phylogenetic analysis, all CTV isolates were grouped into eight clades, which represented as a genogroup, Gr-I to Gr-VIII. The Sikkim isolates used in the present study were segregated into two genogroup, namely Gr-I and Gr-VI. Sikkim CTV isolates SK13, SK20, SK18, SK6, SK11, SK14, SK12 and SK19 showed 98–100 per cent nucleotide identity among them and clustered with previously reported Indian isolate K10 and Florida severe isolate T3. Other isolates viz. SK5, SK7 and SK22 shared 97–99% nucleotide identity within themselves and clustered with Indian isolate K5. These results suggest the presence of genetic diversity among CTV isolates and at least two strains of CTV in Sikkim.

High incidence of citrus tristeza virus in mandarin (Citrus reticulata) in North-East states of India.

Khasi mandarin is one of the most remunerative crop in North Eastern Hills region of India playing a very critical role in the socio-economic upliftment of the people. The orchards of this region were found to express the typical symptoms of citrus tristeza virus (CTV). An extensive survey of the Khasi mandarin orchard for six North Eastern states of India namely Arunachal Pradesh, Meghalaya, Assam, Nagaland, Sikkim and Tripura, was carried out to establish the identity of these viruses using ELISA and PCR/RT-PCR techniques. Out of 300 Khasi mandarin tree samples collected from the six states, 172 were found to be positive for CTV infection by DAS-ELISA indicating 57.33% overall CTV disease incidence. Results revealed presence of CTV in all the surveyed states showing a maximum incidence of 66.00% in Arunachal Pradesh followed by 62.00% in Assam, 60.00% in Meghalaya and Nagaland, 54.00% in Sikkim and 42.33% in Tripura. Higher CTV concentration was recorded in the age group>15years (69.09%) followed by 10-15 (57%) and 5-10years (43.33%). However, this study, to the best of our knowledge, is the first report for the detection of CTV in Khasi mandarin from Arunachal Pradesh, Nagaland and Tripura and also the first authentic survey of overall disease incidence of CTV Khasi mandarins from the six major mandarin growing North Eastern states of India.

Distribution and population structure of Citrus tristeza virus in Poncirus trifoliata

Citrus tristeza virus (CTV) isolates were detected in Poncirus trifoliata, but few research focused on the distribution and population structure of CTV isolates in this resistant species. In this study, 9 P. trifoliata cv. ‘Zaoyangxiaoye’ seedlings were tested positive for CTV in the field. CTV was detected in petal, stamen, pistil and rootlet tissues from CTV-infected plants by real-time RT-PCR, but the incidence of CTV in young flush and old shoot tissues was 68.9% and 4.4%, respectively. The results also showed the highest relative amount of CTV was detected in pistils, followed by rootlets, stamens, petals and young flush. The lowest CTV accumulation was in old shoots, with a viral load two orders of magnitude lower than in pistils. Furthermore, CTV present at a concentration approximately 5 to 10 times higher in petals than in young shoots. These results indicated that the floral parts and rootlets of Zaoyangxiaoye trifoliate orange were more susceptible to CTV accumulation than shoots. Furthermore, T36, T30 and VT genotypes were detected in all of CTV-infected petal, stamen, pistil, rootlet and young flush tissues, but only VT genotype was found in old shoots. Further analysis by specific real-time RT-PCR suggested that the relative amount of VT genotype was most abundant in all of tissue samples, followed by the T30 and T36 genotypes.

Identification and distribution of aphid vectors spreading Citrus tristeza virus in Darjeeling hills and Dooars of India

Darjeeling hills and Dooars of West Bengal (India) are well known for production of mandarin orange and lime. Citrus tristeza virus (CTV) is wide spread in this region. Role of insect vectors in spreading CTV in this region has not been studied so far. Therefore, a study on identification of insect vectors along with their temporal and spatial distribution was undertaken. Five aphid species were identified from citrus orchards of Darjeeling and Dooars viz. Toxoptera citricida, T. aurantii, Aphis gossypii, Myzus persicae and Brachycaudus helichrysi. T. citricida was found predominant in the orchards of lower altitude and was responsible for maximum spread of CTV. T. aurantii was dominant in the citrus orchards at high altitude (>500 m). Incidence of CTV was higher in the orchards where T. citricida was present either alone or with other species. Under caged conditions, T. citricida was more efficient to transmit CTV than the other aphid species. Occurrences of all aphid species were highly influenced by the advent of new flushes.

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.

Prevalence ofCitrus tristeza virusin North Eastern region of India and molecular characterization of its isolates

Incidence of Citrus tristeza virus (CTV) in commercially important citrus species; Khasi mandarin (Citrus reticulata), Assam lemon (Citrus limon) and rough lemon (Citrus jambhiri) in Assam, Meghalaya, Arunachal Pradesh and Nagaland of Northeast (NE) regions of India was studied using double antibody sandwich ELISA (DAS-ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Incidence of CTV disease varied with regions and species of citrus. Incidence was more in Assam lemon than in Khasi mandarin and rough lemon. The viral inoculum was found to be more in Assam and rough lemon than in Khasi mandarin as indicated by higher titre of infected samples. Eight CTV isolates from this region were characterized based on sequencing of coat protein (CP) gene. Sequence analysis showed that CP gene of the present CTV isolates shared 95–99% identity among them. Seven CTV isolates AP3, AP5, JRT1, JRT5, N1, N15 and TK1 were found to be phylogenetically related to each other forming one genogroup and isolate TK 5 was different from other isolates forming another group. Although, recombination events are weak, recombination-detecting program RDP3 showed that majorities of the present isolates are putative recombinants.

Molecular and biological evidence for a severe seedling yellows strain of Citrus tristeza virus spreading in southern Italy

Citrus tristeza virus (CTV) outbreaks have been reported in the main citrus-growing regions of Italy in the past 10 years. In some areas where eradication efforts failed to suppress spread, high CTV incidence is now observed. Recently, potentially severe CTV strains were detected in Calabria (southern Italy), one of the major citrus-growing area. As a result, investigations of the virulence and molecular features of CTV populations spreading in this region were undertaken. Virus was detected by enzyme-linked immunosorbent assay (ELISA) using a broad spectrum polyclonal antiserum, and was differentiated into potential virulent categories with the severe-strain discriminating monoclonal antibody MCA13. Isolate genotyping was conducted using reverse-transcription polymerase chain reaction (RT-PCR) with multiple molecular markers (MMM), single-strand conformation polymorphism (SSCP) analysis of the amplicons from the genes coding for the coat protein (CP) p25 and the non-structural p20 protein as well as sequence analysis. Based on the serological reactivity, the isolates were differentiated in two distinct serogroups: MCA13-reactive and MCA13 non-reactive. Similarly, based on the molecular profile, the isolates were grouped in two genetically distinct phylogenetic clusters, and associated either with a T30-like or with a T3-like genotype. These data were related to the results of biological indexing on standard indicator plants, which distinguished isolates causing mild or severe seedling yellow reactions. The study has demonstrated the presence of MCA13-reactive isolates associated with a T3-like genotype and causing severe seedling yellows in sour orange, grapefruit and lemon seedlings, and stem pitting in Mexican lime.

Current status of Citrus tristeza virus incidence and its spatial distribution in citrus growing geographical zones of India

Citrus tristeza virus (CTV), a member of genus Closterovirus, is an important pathogen which has destroyed more than one million citrus trees in India till date. Survey was conducted in commercial Citrus sp. covering all the citrus growing geographical zones of India. For detection of CTV and estimation of disease incidence, direct antigen coated- enzyme linked immuno-sorbent assay (DAC-ELISA), reverse transcription- polymerase chain reaction and nucleotide sequence analyses were employed. The overall CTV incidence of 26.3% in Vidarbha region, 47.1-56.0% in Northeast (Asom, Meghalaya, Sikkim and the Darjeeling hills), 36-50% in South (Andhra Pradesh and Karnataka) and 16-60% in North-Northwest (Uttarakhand, Delhi and Punjab) were reported. CTV titre in infected tree samples of different locations was measured by ELISA . The high titre was observed in Kagzilime (C. aurantifolia) and sweet orange (C. sinensis) but low in mandarin (C. reticulata). Nucleotide sequence of 5’ORF1a fragment of CTV genome of nine representative CTV isolates, Kat-8, RA-3, B-6, Dh-1, An-9, TP-4, Pant-3, MU-2 and As-2 from all the citrus growing regions surveyed were analysed. The present Indian isolates showed 78-98% identities among them, and placed in different clusters in phylogenetic analysis. This is for the first time an overall incidence of CTV and its spatial distribution in citrus growing geographical zones of India were reported. Findings presented in this study will help in understanding epidemiology and formulating molecular based management strategy.

Present status of Citrus tristeza virus infecting Citrus spp. in Darjeeling hills and its detection in different plant parts

Darjeeling hills of West Bengal (India) are well known for the production of mandarin orange (Citrus reticulata). The spread of Citrus tristeza virus (CTV) in this area has threatened the cultivation of citrus. Therefore, a study of the incidence and distribution of CTV in Darjeeling hills was undertaken and a geographical map was prepared. In DAS-ELISA, a high titre of CTV was recorded in the tender shoot bark, leaf petiole and mid-rib of seven Citrus spp. Three citrus species, viz., pomelo, trifoliate and kumquat, were found to be free of CTV infection when tested in double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription polymerase chain reaction (RT-PCR).

DETECTION OF CITRUS TRISTEZA VIRUS AND CITRUS VIROID SPECIES IN JAMAICA

Field surveys for Citrus tristeza virus (CTV) and citrus viroids were conducted in the six major citrus producing regions across Jamaica. A total of 57 samples were tested by double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) for CTV and reverse transcription polymerase chain reaction (RT-PCR) for CTV and citrus viroids. DAS-ELISA revealed an overall CTV infection of 30-100%, with incidences of severe strains at 50-100%. RT-PCR detected CTV in 10-75% of the samples, while 9-67% were infected with at least two viroid species. Citrus viroids of four groups were prevalent including Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the causal agents of exocortis and cachexia diseases, respectively. Overall, CTV incidence was higher than that of the citrus viroids, except in one region where the incidence of CEVd was higher. Biological indexing confirmed the presence of viroids and mild, decline and stem pitting CTV strains; additionally, mixed infections with CTV strains (13%) and CTV with viroid infections (53%) were observed. Nucleotide sequencing of the CTV coat protein gene and viroid genome sequences isolated from these samples indicated high identities among some of the Jamaican isolates and previously reported CTV and citrus viroid isolates from Central and North America and Eurasia (90-100%, 39-100%, respectively). The findings suggest the need for appropriate certification programmes and management practices to mitigate the impact of both pathogens on the citrus industry.

Prevalence of Citrus tristeza virus in Mandarin of Sikkim Himalayan Region

The assessment of Citrus tristeza virus incidence in mandarin of Sikkim, involving sampling techniques, was estimated by DAS-ELISA. Mandarin orchards had high CTV incidence (46.32%), however, differential prevalence with regard to age of plant and location was observed. The CTV prevalence was relatively high in older orchards (51.01%) than that of younger ones (40.80%). Under all the plant age groups, south district had the highest CTV incidence (52.50%) and east district had the lowest (37.71%). The spatial distribution of CTV in plants indicates high concentration in twig followed by leaf tissue, however, stem had relatively less concentration. High aphid infestation was observed in all mandarin growing groves with the maximum in south district and minimum in east district. Taxoptera citricida was the predominating aphid species followed by T. aurantii, however, Aphis spp population was significantly less. Aphid infestation and CTV prevalence were positively and significantly correlated.

Preliminary Observations on the Phytosanitary Status of the Croatian Satsuma Mandarin (Citrus unshiu Marc.) Collection

Preliminary surveys of citrus field trees in Croatia showed their unsatisfactory sanitary status especially with regard to the presence of Citrus tristeza virus (CTV) even though symptoms in the field are mostly absent. The highest CTV incidence was recorded in the Satsuma mandarin (Citrus unshiu Marc), commercially the most important citrus in the country. The Satsuma collection orchard was founded in the year 2005 at the Institute for Adriatic Crops and Karst Reclamation. It encompasses 12 mandarin cultivars and 51 Satsuma clones from different sources. With the aim of assessing the phytosanitary status of Satsuma collection, biological indexing of all 12 cultivars for principal virus and virus-like diseases was performed. Seven citrus bioindicators were used ; Mexican lime, Madame Vinous and Pineapple sweet oranges, Sicilian sour orange, CRC grapefruit, Troyer and Carrizo citranges. Herbaceous hosts (white sesame, red kidney bean and Chenopodium quinoa) were also used for cool-climate pathogen detection. Serological tests (ELISA and DTBIA) were performed for CTV and Citrus psorosis virus (CPsV) directly on 18 representative clones and on Mexican lime and Madame Vinous indicators. CPsV was not detected by serology while only 2 clones were CTV-negative, both in biological and laboratory tests. Furthermore, the most severe CTV strains displaying stem-pitting and seedling yellows in grapefruit and sweet oranges were found in 4 tested clones. No biological indication of other disease presence in the collection has been found so far. Further testing of the collection Satsumas is under way, as well as the sanitation of the most important cultivars.

Desempeño de la chironja (clon 2-4) injertada en cinco patrones luego de ocho años creciendo en dos zonas en Puerto Rico.

El clon 2-4 de chironja (Citrus sinensis x Citrus paradisiaca) fue evaluado durante los primeros ocho años de crecimiento en las localidades de Isabela y Corozal, Puerto Rico. Los árboles se injertaron en los patrones 'Swingle citrumelo' (Citrus paradisi x Poncirus trifoliata), 'Naronja' (Citrus paradisi x Citrus aurantium), en las mandarinas 'Cleopatra' y 'Sun Chu Sha' (Citrus reticulata), y en el híbrido HRS 812 (Sunki x Beneck trifoliate orange). En ambas localidades, los árboles en la combinación Swingle-chironja murieron durante el primer año de injertados, posiblemente por incompatibilidad de injerto patrón. El patrón HRS 812 resultó ser uno de los mejores productores, aún cuando estuvo infectado con la cepa severa del virus de la tristeza de los cítricos (VTC) en ambas localidades. Aunque las frutas en este patrón fueron de tamaño pequeño, la cantidad de jugo que produjo fue significativamente superior a la de los demás patrones. Los árboles de Chironja injertados sobre Naronja fueron afectados por el VTC; un 38% murieron en Isabela y 25% en Corozal. Sin embargo, los árboles en este patrón crecieron vigorosos y resultaron buenos productores con frutas de buen tamaño y calidad moderada. La alta incidencia de VTC y Phytophthora spp. fue un factor limitante para los patrones susceptibles a estas enfermedades, por lo que hubo una alta mortandad de los árboles.

First Report of Citrus leaf blotch virus in New Zealand.

Despite a high incidence of Citrus tristeza virus (CTV) in citrus in New Zealand, viral diseases have had only a minor impact on the New Zealand citrus industry, largely because of the use of Poncirus trifoliata and hybrid rootstocks derived from this. In August of 2007, a PCR-based survey for seven citrus viruses was conducted on 104 commercial orchard trees that represented a range of Citrus scion species, as well as P. trifoliata and P. trifoliata × Citrus sinensis rootstock, grown from imported and local budwood or from seed in the case of rootstocks, from the citrus-growing regions of Kerikeri, Tauranga, and Gisborne. Total RNA was extracted from young, green bark and leaf tissue from each source. Using a primer pair amplifying a 425-bp region of the Citrus leaf blotch virus (CLBV) coat protein gene (sense: 5'-AGCCATAGTTGAACCATTCCTC-3' and antisense: 5'-GCAGATCATTCACCACATGC-3'), 26 (25%) of the plant samples yielded a DNA fragment of the size expected for CLBV, including 6 of 21 C. sinensis, 1 of 2 C. limon, 3 of 10 C. unshiu, and 5 of 7 C. paradisi scion samples and 5 of 9 P. trifoliata and 6 of 9 P. trifoliata × C. sinensis rootstock samples. Identification of CLBV (an unclassified member of the family Flexiviridae) was confirmed by amplification of a second region of the genome of 1,045 bp spanning the ORF2/ORF3 domains (sense: 5'-ATGAAAAGCCAGTTATGCACCA-3' and antisense: 5'-CTCAGCATTCCCAGGAATAACC-3'). A subset of the PCR products for the CP and ORF2/ORF3 fragments were sequenced for analysis (GenBank Accession nos. EU670243-EU670251 and EU862284-EU862288). Sequences from all CLBV isolates showed 97 to 99% nucleotide identity with the CLBV reference isolate SRA-153 (GenBank Accession No. AF318061) (1) in the coat protein fragment and 98 to 99% nucleotide identity for the partial ORF2/ORF3 fragment. The bud-union crease symptom reported to be caused by CLBV was observed only on one C. paradisi scion on P. trifoliata rootstock, while the remaining samples were either asymptomatic or symptoms were attributed to co-infection with CTV. Since CLBV-infected plants were found from all major growing regions, it is apparent that CLBV is widespread in New Zealand, although it is not known where in this country it may have originated. Reference: (1) M. C. Vives et al. Virology 287:225, 2000.

Citrus tristeza virus: a pathogen that changed the course of the citrus industry

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures.

Incidence, Transmissibility, and Genotype Analysis of Citrus tristeza virus (CTV) Isolates from CTV Eradicative and Noneradicative Districts in Central California.

Growers in 45% (44,100 ha) of the citrus acreage in California stopped eradicating Citrus tristeza virus (CTV)-infected trees from their fields in 1995-96. The impact of leaving infected trees on the rate of CTV spread was determined by comparing temporal incidence of CTV in plots in Strathmore, Tulare County without eradication with incidence in a plot in McFarland, Kern County with eradication. From 1997 to 2003, CTV incidence in the Strathmore plots ranged from 6 to 42%, with annual spread rates from 1.6 to 3.6%. CTV incidence in the McFarland plot increased from 0 to 5% between 2001 and 2003 before infected trees were removed. Using a subplot hierarchical bulk sampling method, virus incidence over a 3-year period in a 6.5 km2 area near McFarland was estimated to range from 0.09 to 0.69%, which indicated that CTV suppression was still being achieved in this area. Vector tests using the cotton aphid, Aphis gossypii, identified highly transmissible isolates (30 to 61% transmission rate) and a larger proportion of highly transmissible isolates were found in the McFarland plots. Thirty-six CTV isolates from recently infected plot trees were obtained and analyzed. None of these isolates reacted with monoclonal antibody MCA13 that detects presumptive CTV severe strains. Molecular analysis using polymerase chain reaction and sequence-specific primers showed that all isolates had a genotype identical to the T30 mild isolate from Florida.