On the spatial distribution of citrus tristeza virus disease

The spatial spread of citrus tristeza virus (CTV) disease was studied using data from two infected groves. Statistical analysis indicated a spatial grouping of infected trees. A gamma distribution was fitted to the empirical distribution of the distances between nearest infected neighbors. The findings suggest that, after locating a newly infected tree, additional inspections to detect CTV should be carried out mainly on trees within 14 m of that tree.     

Survey methods for assessment of citrus tristeza virus incidence

ABSTRACT The assessment of citrus tristeza virus incidence by sampling involves laboratory testing by enzyme-linked immunosorbent assay of leaf material collected in the field. Using field data and computer simulation, methods of field data collection were compared. One method was similar to that used by the Central California Tristeza Eradication Agency, in which 4 to 6% of the trees in a planting block are sampled and material from each tree sampled is assayed separately. This method was compared with an alternative method in which about 25% of the trees in a block are sampled, and material from groups of four trees is bulked and assayed together. Our comparative study indicated that the latter method results in increased accuracy and precision of estimates of citrus tristeza virus incidence without increasing unduly the number of laboratory assays required.     

Biological characterization of citrus tristeza virus isolates by in vitro tissue cultures

Stem segments from Mexican lime, sweet orange, grapefruit, Citrus excelsa and alemow, infected with five citrus tristeza virus (CTV) isolates, were cultured in vitro . Regeneration of roots and shoots were modified as a result of infection. The effect of CTV on the morphogenesis of stem segments cultured in vitro depended on the CTV isolate and the plant host, and showed a correlation with the in vivo effects observed in biological indexing. Evaluation of the morphogenic response of stem segments of Mexican lime and grapefruit can be used as an additional tool for the biological characterization of CTV isolates. The symptoms on sweet orange plants obtained from regenerated shoots indicated that CTV is unevenly distributed in the host plant cells and that the regeneration process may be utilized as a tool to separate strains from complex field isolates.     

Biological diversity of citrus tristeza virus (CTV) isolates in Spain

A survey of citrus tristeza virus (CTV) isolates was carried out in most citrus-growing areas in Spain. Twenty-two isolates were selected by geographical origin, cultivar of source tree, and symptoms observed on the host or in preliminary tests, and were biologically characterized.
A wide range of variation in transmissibility by aphids and symptom intensity on nine different indicator species or scion-rootstock combinations was observed among CTV isolates. Mexican lime. Citrus macrophylla , and to a lesser extent citron were the most useful hosts for characterizing these isolates, and leaf symptoms and stem pitting were the most discriminating traits. Positive correlation was observed between symptoms induced on Mexican lime and C. macrophylla , but not between the symptoms induced on these indicators under greenhouse conditions and the homologous symptoms on plants grown in the screenhouse. Some of the traits studied enabled us to establish relatively well-defined groups of isolates, but in most cases a continuous range of variation was obtained and no clear group could be defined.     

Variability among Spanish citrus tristeza virus isolates revealed by double-stranded RNA analysis

A survey for citrus tristeza virus (CTV) strains, based on double-stranded RNA (dsRNA) analysis, was carried out in five locations on the eastern citrus-growing area of Spain. CTV was recovered from 137 trees of different ages, citrus species and varieties, sampled in 53 orchards. The best months for dsRNA recovery were April, May, September, October, and November, and the highest dsRNA yield was obtained from sweet orange cultivars. Sixteen dsRNA profiles differing by the number and/or position of subgenomic bands were detected. One of these profiles was detected in more than half the trees analysed. Maximum diversity of dsRNA patterns was found in the location with the oldest citrus orchards and the highest CTV incidence (Alzira-Carcaixent). In many instances, several dsRNA profiles were detected in neighbouring trees of the same orchard, notably in Alzira-Carcaixent, where 70% of the plots sampled contained more than one profile. The possible causes for the diversity of CTV isolates found in this specific area are discussed.     

Epidemiological and economic models for spread and control of citrus tristeza virus disease

Citrus tristeza virus (CTV) causes a most destructive citrus disease in many parts of the world, and indications of natural spread were found in Israel in 1970. The strategy for controlling the disease in Israel is based on the eradication of virus-infected trees, detected by test plants or immunological methods. Mathematical models for CTV infection and spread were developed and used to assess the cost-effectiveness of the eradication policy. It was concluded that the discovery-eradication program is economically justified and superior to allowing the disease to progress unchecked.     

Investigation of biological properties, double-stranded RNA patterns and antigen concentration in citrus species infected with citrus tristeza virus

Biological properties and dsRNA patterns of one Cyprus and three Turkish isolates of citrus tristeza virus (CTV) were investigated. In addition, CTV antigen concentration and effect of tissue sampling time from naturally infected Shamouti sweet orange trees grown in the field of Icel Province, Turkey, were also determined. The Cyprus isolate showed vein clearing symptoms on grapefruit, ‘Madam Vinous’ and Mexican lime and stem pitting symptoms on Mexican lime. The three Turkish isolates showed only vein clearing symptoms on Mexican lime. All four isolates showed a full-length major double-stranded RNA (dsRNA) band of 13.3 × 10⁶ Da mol. wt in extracts from infected Madam Vinous sweet orange trees, and major or minor dsRNA bands with 2.0. 0.8 and 0.5 × 10⁶ mol.wt. All seven different citrus varieties inoculated with the Igdir (D) strain contained full-length dsRNA. The additional two dsRNA of 0.8 and 0.5 × 10⁶ mol.wt were also detected as clearly as full-length dsRNA in these hosts, but were weaker inCitrus exelsa and ‘Interdonat’ lemon. Madam Vinous, rough lemon and Mexican lime were the best hosts for dsRNA analysis. ELISA values were highest in April (OD_405nm =0.476), decreased steadily until August, and then increased gradually through December. ELISA values were lowest in July and August (OD_405nm =0.157 and 0.141, respectively). dsRNA recovery from a field tree infected with isolate Igdir D was good in March, April and May and poor in January and February. No dsRNA band was detected in August or September. http://www.phytoparasitica.org posting July 9, 2002.     

Molecular variability of the 5'- and 3'-terminal regions of citrus tristeza virus RNA

ABSTRACT Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.     

6种柑橘类植物对柑橘衰退病毒分离株TR-L514变异的影响

 柑橘衰退病毒(Citrus tristeza virus,CTV)存在复杂的株系分化现象,运用弱毒株交叉保护防治柑橘衰退病时需了解不同类型柑橘对CTV构成的影响。作者将CTV分离株TR-L514嫁接接种到6类柑橘植物上获得了18个亚分离株,并对这18个亚分离株和TR-L514进行了指示植物鉴定,p25基因的限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,p23基因的序列比较。结果表明,CTV株系对不同柑橘类植株的适应性存在差异。TR-L514嫁接到不同柑橘类植株其构成会发生变化,在甜橙上可以同时检测出p25//HinfⅠ RFLP第1和6组群,并具有比在其它4类柑橘上更为复杂的SSCP谱型构成,因此甜橙可能更适宜于CTV的增殖。TR-L514及18个亚分离株的p23基因序列差异可能与不同类柑橘植株的适应性有关。     

广西柑橘衰退病和黄脉病调查及其病原病毒的遗传多样性分析

为明确柑橘衰退病毒(citrus tristeza virus, CTV)和柑橘黄脉病毒(citrus yellow vein clearing virus, CYVCV)在广西柑橘上的发生?分布及其遗传变异情况, 于2020年至2021年对百色?北海?崇左?贵港?桂林?河池?贺州?来宾?柳州?南宁?梧州和玉林等12个柑橘产区进行了病毒病调查?采用RT-PCR对采集样品进行了病毒检测, 并基于病毒分离物外壳蛋白(coat protein, CP)基因的核苷酸序列进行比对分析, 构建系统发育树?结果表明:采集的737份柑橘样品中, CTV的检出率为20.62%, CYVCV检出率为18.32%, CTV的检出率略高于CYVCV?病毒复合侵染的现象在采集的柑橘样品中普遍存在, CTV和CYVCV复合侵染率高达34.50%?对RT-PCR产物测序共获得12个CTV分离物和6个CYVCV分离物的CP基因序列?遗传多样性分析发现, CTV和CYVCV的CP基因序列都较保守, CTV分离物的遗传进化与地理来源?寄主来源均没有明显相关性, 但CYVCV分离物的遗传进化与地理位置具有相关性, 而与寄主来源无明显相关性?上述研究结果可为深入了解CTV和CYVCV在广西的流行情况以及柑橘病毒病的检疫和防控提供参考?     

Examination of the effect of aphid vector population composition on the spatial dynamics of citrus tristeza virus spread by stochastic modeling

ABSTRACT Aphid vector species population composition is known to affect the spatial patterns of citrus tristeza virus (CTV) and the changes in these patterns over time. However, the biological processes that are associated with virus spread have not been well defined. The spatiotemporal dynamics of CTV were examined using data collected from research plots in the Dominican Republic and Costa Rica, where the brown citrus aphid (BCA), Toxoptera citricida, was the predominant species, and in Florida, where the BCA was absent and the melon aphid, Aphis gossypii, was the predominant vector. Data were analyzed using a spatiotemporal stochastic model for disease spread, and parameter values were evaluated using Markov chain Monte Carlo stochastic integration methods. Where the melon aphid was the dominant species, the model parameter likelihood values supported the hypothesis that the disease was spread through a combination of random background transmission (transmission originating from inoculum sources outside the plot) and a local interaction (transmission from inoculum sources within the plot) operating over short distances. Conversely, when BCA was present, results often suggested a local short-range transmission interaction that was not restricted to nearest-neighbor interactions and that the presence of background infection was not necessary to explain the observations.     

Spatiotemporal dynamics of Citrus tristeza virus in Cuba

The spatiotemporal dissemination of Citrus tristeza virus (CTV) was evaluated by DASI-ELISA in orange and grapefruit fields of six citrus producing regions in Cuba, and aphid populations were evaluated in two selected areas. The aphid species found in these areas were Toxoptera citricida (the most efficient vector of CTV), T. aurantii and Aphis spiraecola . A logistic model was the most appropriate to explain the temporal increase in the proportion of CTV-infected plants for almost all the fields. Although nearly all areas showed an increase in this proportion, there were regions of low CTV spread, which was slower in grapefruit than in orange fields. 2DCORR analysis indicated spatial dependence among immediately adjacent trees, higher in the within-row direction than in the across-row direction. Aggregation was also detected by dispersion index and binary power law fitting within quadrats of all sizes. For most fields, autocorrelation analysis showed a significant edge effect and spatial dependence among infected plants of different sub-areas. Based on these results a new tristeza management strategy was proposed for each region of the country.     

Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

First detection of a virulent strain of Citrus tristeza virus (Closteroviridae) in a citrus orchard of Chlef Valley (Algeria)

A large‐scale survey of Citrus tristeza virus (CTV) was carried out from 2016 to 2018 in the Chlef Valley, one of the main citrus growing areas in Algeria. In this study a total of 1680 citrus trees from 93 commercial orchards were sampled. The collected samples were tested by direct tissue blot immunoassay analysis and by the double antibody sandwich enzyme‐linked immunosorbent assay technique, and 54 trees were identified as being infected with CTV. This result confirmed that 54 trees were infected by the virus, corresponding to an infection rate of 3.21% throughout the studied area. Five of these local CTV sources were chosen for further molecular investigations to determine the genotype associated with the CTV isolates now spreading in the Chlef area. Characterization with multiple molecular markers showed the presence of the T30 and VT genotypes. This result allowed confirmation of the presence of a virulent strain belonging to the VT genotype. The other CTV isolates were similar to those from the Mitidja region, which showed 99% nucleotide identity with the Spanish mild CTV isolate. This early finding of a strain belonging to the VT genotype is an issue for Algerian citrus producers and needs rapid actions to be taken by the National phytosanitary services, extending the surveillance to other citrus production regions and uprooting the infected trees.     

Contribution of uneven distribution of genomic RNA variants of citrus tristeza virus (CTV) within the plant to changes in the viral population following aphid transmission

The population of genomic RNA sequence variants of citrus tristeza virus (CTV) isolates was characterized by single-strand conformation polymorphism (SSCP) analysis of complementary DNA (cDNA) of the genes p18 and p20. Comparison of field and aphid-transmitted isolates showed that aphid transmission frequently altered the single-strand conformation polymorphism (SSCP) pattern of both genes, indicating changes in the population of genomic RNA variants. SSCP analysis of the cDNA of RNA extracted from small pieces of tissue sampled at different sites of the same plant sometimes yielded different patterns, indicating uneven distribution of the genomic RNA variants within the infected plant. Different SSCP patterns were also obtained when the RNA extracted from individual aphids probing in the same infected leaf was used as a template. Uneven distribution of the genomic RNA variants within the infected plant and sorting of some of these variants by individual aphids probably contribute to changes observed in the CTV population following aphid transmission.     

Separation and interference of strains from a citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis

Separation of strains of citrus tristeza virus (CTV), differentiated by their double-stranded RNA (dsRNA) profiles, was obtained by graft-inoculating citron plants from a Mexican lime that had been recently aphid- or graft-inoculated with a mild CTV isolate (T-385). Up to 24 sub-isolates with differing dsRNA profiles were obtained from the aphid-inoculated lime. Some of these sub-isolates induced stronger symptoms in several citrus species than the original T-385 isolate. One sub-isolate, T-385-33, was mild in Mexican lime, but induced stem pitting on sweet orange. Inoculation of this isolate on Mexican lime, sour orange and Eureka lemon induced mild or no symptoms when inoculum was taken from citron, but very severe symptoms when the inoculum was from sweet orange. Mexican lime and sweet orange plants co-inoculated with T-385-33 from sweet orange in combination with the other 23 sub-isolates showed mild symptoms. The results obtained suggest that there is natural cross-protection among sub-isolates in the original T-385 isolate.     

柑桔衰退病毒弱毒株及其筛选方法

不同研究方法揭示了柑桔衰退病毒存在着复杂的株系分化现象。本文用分子生物学技术与生物学鉴定相结 合的方法区别柑桔衰退病毒强弱毒株,利用其弱毒株交叉保护机理为筛选有保护作用的弱毒株提供了强有力的手 段。     

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.