Increase and Patterns of Spread of Citrus Tristeza Virus Infections in Costa Rica and the Dominican Republic in the Presence of the Brown Citrus Aphid, Toxoptera citricida

ABSTRACT Citrus tristeza virus (CTV) was monitored for 4 years by monoclonal antibody probes via enzyme-linked immunosorbent assay in four citrus orchards in northern Costa Rica and four orchards in the Dominican Republic following the introduction of the brown citrus aphid, Toxoptera citricida. The Gompertz nonlinear model was selected as the most appropriate in most cases to describe temporal increase of CTV. Ordinary runs analysis for association of CTV-positive trees failed to show a spatial relationship of virus status among immediately adjacent trees within or across rows. The beta-binomial index of dispersion for various quadrat sizes suggested aggregations of CTV-positive trees for all plots within the quadrat sizes tested. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to four lag distances; however, significant lag positions discontinuous from the main proximity pattern were rare. Some asymmetry was also detected for some spatial autocorrelation proximity patterns. These results were interpreted to mean that, although CTV-positive trees did not often influence immediately adjacent trees, virus transmission was common within a local area of influence that extended two to eight trees in all directions. Where asymmetry was indicated, this area of influence was somewhat elliptical. The spatial and temporal analyses gave some insight into possible underlying processes of CTV spread in the presence of T. citricida and suggested CTV spread was predominantly to trees within a local area. Patterns of longer-distance spread were not detected within the confines of the plot sizes tested. Longer-distance spread probably exists, but may well be of a complexity beyond the detection ability of the spatial analysis methods employed, or perhaps is on a scale larger than the dimensions of the plots studied.     

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.     

Molecular characterization of Citrus yellow mosaic badnavirus (CMBV) isolates revealed the presence of two distinct strains infecting citrus in India

Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etiologic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur isolate formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.     

Detection and characterization of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> stem pitting isolates in Jamaica

An island wide survey for Citrus tristeza virus (CTV) in citrus orchards across Jamaica (13 regions) was conducted over 2 years. Trees (1, 885) showing virus-like symptoms as well as asymptomatic trees were randomly sampled for testing by ELISA and 55 samples from the 6 major citrus growing regions were graft inoculated on indicator plants. Most samples (74%) reacted to polyclonal antibodies against CTV in ELISA, while 20% were positive in tests using monoclonal antibodies specific to severe CTV strains. Samples collected from the 6 major citrus growing regions produced vein clearing and stem pitting symptoms on Mexican lime indicator plants (87%). In addition, stem pitting symptoms were induced on Duncan grapefruit, sweet orange, sour orange or sweet orange grafted on sour orange. Nucleotide sequencing of the coat protein gene sequences isolated from these samples indicated high identities (88 to 95.5%) among the Jamaican isolates and previously reported stem pitting strains from Central and North America and Eurasia (88 to 100%). The results suggest a shared ancestry with isolates from other geographical locations, rather than geographical speciation, and presumably separate CTV introductions into Jamaica.     

Spatial patterns of leprosis and its mite vector in commercial citrus groves in Brazil

In order to describe the dissemination of Citrus leprosis virus (CiLV) and Brevipalpus phoenicis , the Tenuipalpidae mite which transmits it, as well as to generate data for future development of better sampling and disease control procedures, analyses were conducted at three levels of spatial hierarchy. Over 100 distribution maps of mite-infested plants and plants with CiLV symptoms were constructed after evaluation of 174 sweet orange commercial grove blocks from four citrus regions of São Paulo State, Brazil. Spatial correlation between maps of mite-infested plants and those with CiLV symptoms was very low and mostly not significant. Spatial dependency between adjacent plants was incipient as ordinary runs analysis showed that percentages of aggregated sequences within or across rows were very low for both mite-infested and diseased plants. Index of dispersion ( D ) values for all quadrat sizes suggested aggregation of plants with symptoms within quadrats, but much less aggregation for mite-infested plants. Values of log( A ) and b were higher than 0 and 1, respectively, indicating a general and significant aggregation of infested/diseased plants inside quadrats. However, the degree of aggregation of plants with symptoms was higher than that of infested plants. Aggregation in each grove was positively correlated to the incidence of infested/diseased plants. Spatial autocorrelation also showed a higher strength of aggregation for plants with symptoms than for infested ones. This is the first time that spatial patterns of leprosis and B. phoenicis have been described.     

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.     

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.     

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.     

Spatial patterns of the Citrus leprosis virus and its associated mite vector in systems without intervention

Leprosis is caused by the Citrus leprosis virus cytoplasmic type and is vectored by the mite Brevipalpus yothersi. Miticide applications, which cost $54 million annually, are based on inspection for the presence of mites. The aim of the present study was to characterize the spatial patterns of B. yothersi-infested trees and trees with leprosis symptoms for further improvement in sampling and disease control. The presence of mites and the occurrence of leprosis were assessed over two years in 1160 Valencia trees and 720 Natal trees in a commercial sweet orange grove in Sao Paulo State, Brazil. To assess the natural growth and dispersal of mites and leprosis, mite populations were not controlled during the experimental period. Maps of mite-infested trees and trees with leprosis symptoms were analysed at three different levels of spatial hierarchy using complementary methods, i.e. among adjacent trees within and across rows, within quadrats, and the strength and orientation of aggregation among quadrats. The study showed that the spatial patterns of virus-infected and mite-infested trees were different, with a strong aggregation pattern of trees with leprosis symptoms that increased over time. Conversely, the spatial pattern of B. yothersi showed randomness or weak aggregation at all three spatial hierarchical levels. Disease incidence increased steadily in plots of both cultivars, unlike in mite-infested trees where incidence fluctuated over time. These results have important implications for the development of better management strategies for leprosis. Sampling methods and action thresholds for mite control should consider primary disease inoculum in addition to the incidence of mites.     

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.     

Evidence for resistance to Citrus tristeza virus in pomelo (Citrus maxima Merr.) grown in Darjeeling and Sikkim hills of India

Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.     

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.     

枸头橙种子中柑桔衰退-茎陷点病毒的鉴定

 采用双抗体夹心-酶联免疫吸附测定试验(DAS-ELISA)对枸头橙种子中柑桔衰退-茎陷点病毒的带毒情况进行了鉴定。结果表明,从严重感染茎陷点病的枸头橙罹病树采集的种子可检测到柑桔衰退病毒(CTV)。种子带毒量以内种皮为较高,剥皮种子次之,外种皮呈阴性。种子在4℃冰箱内贮藏1~2年后仍带毒,但带毒量有所下降。对带毒种子繁殖的实生苗及嫁接在实生苗上的墨西哥来檬的检测结果,有部分呈阳性,种植在防虫隔离网室内的100余株2年生枸头橙实生苗,用无毒珠心系的墨西哥来檬芽嫁接进行生物鉴定,经2年观察未发现症状     

The influence of citrus rootstocks on the relationship between the mite Brevipalpus phoenicis and citrus leprosis disease

BACKGROUND: Leprosis is one of the most serious citrus plant diseases. Leprosis‐affected plants, especially sweet orange [Citrus sinensis (L.) Osbeck], which is the most widely cultivated citrus fruit worldwide, show reduced photosynthetic capacity and severe defoliation. The aim was to evaluate the relationship between the Brevipalpus phoenicis (Geijskes) vector mite and citrus leprosis disease in Pera sweet orange plants grafted on different rootstocks. Data were analysed using numerical classification and conventional statistical analysis (ANOVA). RESULTS: Both viruliferous and non‐viruliferous B. phoenicis populations increased in number on plants maintained at low soil water content. Among the evaluated rootstocks, Sunki mandarin proved least favourable for mite population increase. Furthermore, the viruliferous mite population increased more rapidly than the non‐viruliferous mite population. CONCLUSION: The Cleopatra rootstock showed low variability in leaf nitrogen content, low mite number and low leprosis severity, thereby producing the most favourable results. Under appropriate moisture conditions, the Sunki rootstock produced the best results, showing the least severe leprosis. Rangpur lime rootstock grown in soil with varying water content showed the greatest variation in foliar nitrogen content, mite number and severity of leprosis and is considered to be the most difficult rootstock to manage. Copyright © 2012 Society of Chemical Industry     

Inheritance and heritability of resistance to citrus leprosis

ABSTRACT The genetic inheritance of resistance to leprosis, the most important viral disease of citrus in Brazil, was characterized through the phenotypic assessment of 143 hybrids resulting from crosses between tangor 'Murcott' (Citrus sinensis x C. reticulata) and sweet orange 'Pêra' (C. sinensis), considered to be resistant and susceptible to the disease, respectively. All plants were grafted onto Rangpur lime (C. limonia) and inoculated with Citrus leprosis virus, cytoplasmic type through the infestation with viruliferous mites, Brevipalpus phoenicis. The experiments were arranged in a completely randomized block design with 10 replicates. Incidence and severity of the disease in leaves and stems as well as plant growth parameters (plant height and stem diameter) were recorded for 3 years after the infestation with the viruliferous mites. The average values of all variables were analyzed using principal component analysis, discriminant factorial analysis, estimation of the clonal repeatability coefficients, and frequency of the distributions of the average values for each measured variable. The principal component analysis resulted in the identification of at least two groups with resistance and susceptibility to leprosis, respectively. About 99% of all hybrids were correctly classified according to the discriminant factorial analysis. The broad-sense heritability coefficients for characteristics associated with incidence and severity of leprosis ranged from 0.88 to 0.96. The data suggest that the inheritance of resistance to leprosis may be controlled by only a few genes.     

Selective transmission of the seedling yellows-type genotypic variants of Citrus tristeza virus by Aphis gossypii in northern Iran

Citrus tristeza virus (CTV) has existed in northern Iran for more than five decades. The long-time interaction of different virus genotypes with Aphis gossypii, as the only aphid vector of CTV in northern Iran, has led to the emergence of highly virulent subpopulations, among others, in the established foci. Here, we studied the population structure of the originally established CTV isolates present in Satsuma mandarin (Citrus unshiu) trees imported from Japan, and subisolates thereof, formed following experimental transmission by A. gossypii, as well as those evolved through natural transmission by this aphid species in the groves. Symptoms of the naturally spread and the experimentally aphid-transmitted isolates were similar to those of the Satsuma CTV source isolates for all indicator plants except for sour orange (Citrus aurantium) and grapefruit (Citrus paradisi), with the aphid-transmitted isolates additionally inducing severe seedling yellows and stunting in these two indicators. Studies on the population heterogeneity of these isolates through comparison of their single-strand conformational polymorphism profiles and nucleotide sequences of the 25 kDa capsid protein gene from the predominant haplotypes, and dot-blot hybridization signals, revealed the presence of two major T36- and SY568- (or NUagA-) like genotypes along with a minor poorly characterized one in the originally infected Satsuma trees; in contrast, only a certain genomic variant having the highest similarity to the isolate SY568 (and NUagA) was predominant both in the naturally infected trees and in those infected experimentally by A. gossypii. It seems that transmission by A. gossypii to sweet orange (Citrus sinensis) has led to the preponderance of the CTV genomic variants inducing severe seedling yellows in northern Iran.     

Reaction of transgenic Citrus sinensis plants to Citrus tristeza virus infection by Toxoptera citricida

Transgenic Citrus sinensis ‘Hamlin’ and ‘Valencia’ plants containing Citrus tristeza virus (CTV)-derived sequences were propagated and inoculated with CTV. For propagation, selected buds from transgenic and non-transgenic control plants were grafted onto C. aurantium and C. limonia rootstock plants. CTV inoculation was performed via viruliferous aphids (Toxoptera citricida), and viral detection post-inoculation was performed through DASI-ELISA or RT-qPCR. After four inoculations, none of the transgenic lines tested showed complete resistance. However, viral multiplication was undetectable in some of the propagated clones. These resistant clones mainly came from transgenic ‘Valencia’ sweet orange plants grafted onto C. limonia rootstock containing the pCTV-CS gene construct. Although the tested viral inoculation method represents natural field infection conditions, the results did not differ significantly from those previously reported when the same transgenic lines were bud-graft inoculated. This finding indicates that the difficulties in producing CTV-resistant transgenic citrus lines may be unrelated to the inoculation method. Transgene expression level was quantified by RT-qPCR analysis and it was not possible to relate transgene mRNA level with resistance to the pathogen.     

Survey Methods for Assessment of Citrus Tristeza Virus Incidence When Toxoptera citricida Is the Predominant Vector

ABSTRACT Citrus tristeza virus (CTV) incidence may be assessed by sampling groups of citrus trees, recording the groups as CTV positive (one or more infected trees) or CTV negative (no infected trees), and then calculating disease incidence at the scale of the individual tree by means of a formula involving incidence at the group scale and the number of trees per group. This procedure works well when the CTV status of a tree can be regarded as independent of the CTV status of other trees in the same group. This is the case when the main vector species is Aphis gossypii and the groups comprise four adjacent trees, because the spatial pattern of CTV incidence at the within-group scale can be regarded as random. However, when the main vector species is Toxoptera citricida, this simple procedure is not appropriate, because the spatial pattern of CTV incidence at the within-group scale cannot be regarded as random. Using field data and computer simulation, an alternative procedure for assessment of CTV incidence when the main vector species is T. citricida was devised and tested. In the alternative procedure, the sampling scheme is operationally identical to that used when the main vector species is A. gossypii, but the calculation of CTV incidence at the scale of the individual tree is based on incidence at the group scale and an effective sample size. The analysis of CTV-incidence data collected from a number of citrus blocks in reasonable geographical and temporal proximity and the use of CTV-detection methods more sensitive than the enzyme-linked immunosorbent assay used here are also discussed.     

Citrus viroid II variants associated with ‘Gummy Bark’ disease

A viroid etiology for citrus gummy bark (CGB) disease of sweet orange is supported by the similarity of symptom expression to cachexia disease of mandarins and tangelos caused by the hop stunt viroid (HSVd) related citrus viroid II (CVd-II), as well as the detection of CVd-II variants in CGB infected Washington navel and Dörtyol sweet orange, a Turkish cultivar. A survey was made of 67 clones of CVd-II related variants recovered from severe CGB symptomatic and non-symptomatic trees of the same cultivars growing in close proximity. Only CVd-IIa, a non-cachexia inducing variant, was found in non-symptomatic Washington navel trees and no CVd-II variants were recovered from the Dörtyol control. CGB infected sources contained a number of CVd-II related variants with the predominant species detected closely related to CVd-IIc, a known cachexia inducing viroid. Biological activity of representactive variants from CGB sources was determined by transmission to citron (Citrus medica) as well as by bioassay on the indexing host for cachexia, Parson's Special mandarin (Citrus reticulata).