Occurrence of Stem-Pitting Strains of Citrus tristeza virus in Croatia

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30′N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field-infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33°C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTV1 and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision. References: (1) G. Nolasco et al. Eur. J. Plant Pathol. 108:293, 2002. (2) A. Roy et al. Arch. Virol. 148:707, 2003. (3) A. Šarić and I. Dulić. Agric. Conspectus Sci. 55:171, 1990. (4) G. Suastika et al. J. Gen. Plant Pathol. 67:73, 2001.

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Production of Antibodies to Citrus Tristeza Virus in Transgenic Citrus

10.32747/1995.7613018.bard ◽

1995 ◽

Author(s):

Richard Lee ◽

Moshe Bar-Joseph ◽

K.S. Derrick ◽

Aliza Vardi ◽

Roland Brlansky ◽

...

Keyword(s):

Citrus Tristeza Virus ◽

Virus Disease ◽

Biological Activities ◽

Cp Gene ◽

Single Domain Antibodies ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Tristeza

Citrus tristeza virus (CTV) is the most important virus disease of citrus in the world. CTV causes death of trees on sour orange rootstock and/or stem pitting of scions regardless of rootstock which results in trees of low vigor, reduced yield with reduction in size and quality of fruit. The purpose of this project was to produce monoclonal antibodies (MABs) to CTV coat protein (CP), develop single domain antibodies (dAbs) or Fab fragments which neutralize the infection by binding to the virus, and to produce transformed plants which express the dAbs. The objectives of this research have been met and putative transgenic tobacco and citrus plants have been developed. These putative transgenic plants are presently undergoing evaluation to determine the level of dAbs expression and to determine their resistance to CTV. Additionally, the CTV genome has been sequenced and the CP gene of several biologically characterized CTV strains molecular characterized. This has indicated a correlation between CP sequence homology and biological activity, and the finding of DI RNAs associated with some CTV strains. Several MABs have been produced which enable broad spectrum identification of CTV strains while other MABs enable differentiation between mild and severe strains. The use of selected MAbs and determination of the CP gene sequence has enabled predictions of biological activities of unknown CTV isolates. The epitopes of two MABs, one reacting selectively with severe CTV strains and the other reacting with all strains, have been characterized at the molecular level.

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Citrus tristeza virus (citrus tristeza).

10.1079/cpc.16705.20210102472 ◽

2021 ◽

Author(s):

Mariano Cambra

Keyword(s):

Citrus Tristeza Virus ◽

Sweet Orange ◽

Salt Content ◽

Fruit Size ◽

Soil Conditions ◽

Sour Orange ◽

Tristeza Virus ◽

Stem Pitting ◽

The Mediterranean Basin ◽

Citrus Tristeza

Abstract CTV is the most economically important virus pathogen of citrus worldwide. About 100 million citrus trees on sour orange have been killed by CTV decline epidemics in Argentina, Brazil, Venezuela, Peru, Florida and California (USA), Israel, Spain, and other locations. With current estimates of approximately 45 million trees on sour orange killed by CTV in Spain (Cambra et al., 2000a; Vidal et al., 2012) from 1935 to date without T. citricida implication. It is estimated that worldwide, mainly in the Mediterranean basin citrus industries, there are over 200 million trees on sour orange rootstock which are at risk to this disease. Sour orange is popular because it produces a vigorous tree with high quality fruit, is adaptable to many soil conditions including high lime and salt content, and has tolerance to many other viruses, viroids and virus-like pathogens, and to Phytophthora. The use of tristeza-tolerant rootstocks often risks losses from other factors. In addition to decline, many severe CTV isolates cause stem pitting diseases of susceptible scion cultivars and these occur even when tolerant rootstocks are used. Stem pitting weakens trees and eventually reduces fruit size, quality and quantity (Marais et al., 1996). Grapefruit and lime are very sensitive to stem pitting. Sweet orange is more tolerant but can be severely affected by some isolates. Mandarin is the most tolerant among the main citrus cultivars against SP isolates.

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Characterization of Citrus tristeza virus Single-Variant Sources in Grapefruit in Greenhouse and Field Trials

Plant Disease ◽

10.1094/pdis-03-16-0391-re ◽

2016 ◽

Vol 100 (11) ◽

pp. 2251-2256 ◽

Cited By ~ 6

Author(s):

Glynnis Cook ◽

Stephanus P. van Vuuren ◽

Johannes H. J. Breytenbach ◽

Chanel Steyn ◽

Johan T. Burger ◽

...

Keyword(s):

Citrus Tristeza Virus ◽

Phenotypic Expression ◽

Field Trials ◽

Cross Protection ◽

Enzyme Linked Immunosorbent Assay ◽

Limiting Factor ◽

Phenotypic Effect ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Tristeza

Citrus tristeza virus (CTV) is endemic to southern Africa and the stem pitting syndrome that it causes was a limiting factor in grapefruit production prior to the introduction of cross-protection in the Citrus Improvement Scheme. This disease mitigation strategy, using various field-derived CTV sources, has significantly extended the productive lifespan of grapefruit orchards in South Africa. CTV commonly occurs as a population of various strains, masking the phenotypic effect of individual strains. Likewise, current South African CTV cross-protection sources are strain mixtures, obscuring an understanding of which strains are influencing cross-protection. The severity of various CTV strains has mostly been assessed on sensitive indicator hosts, but their effect on commercial varieties has seldom been investigated. Single-variant CTV isolates were used to investigate the phenotypic expression of CTV strains in commercial grapefruit varieties as well as CTV indicator hosts. They were biologically characterized for their ability to cause stem pitting and their rate of translocation and titer in the different hosts, monitored by enzyme-linked immunosorbent assay. Complete genome sequences for three CTV strain variants were generated. Isolates of CTV strains VT, T68, RB, and HA16-5 did not induce severe stem pitting in four grapefruit hosts in a glasshouse trial. Viral titers of the strains differed in the grapefruit hosts, but the RB isolate reached a higher titer in the grapefruit hosts compared with the VT, T68, and HA16-5 isolates. Additionally, horticultural assessment of two grapefruit varieties inoculated with the RB isolate in two field trials demonstrated that mild stem pitting did not negatively influence the horticultural performance of the grapefruit trees over an eight-year assessment period. ‘Star Ruby’ trees containing the CTV source GFMS35 showed less stem pitting than trees inoculated with the RB isolate, but had smaller canopy volumes and lower yields than trees containing the RB isolate. This suggests that the influence of CTV sources on tree performance is not limited to the effect of stem pitting.

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Serological Differentiation of the Citrus Tristeza Virus Isolates Causing Stem Pitting in Sweet Orange

Plant Disease ◽

10.1094/pdis.1998.82.11.1276 ◽

1998 ◽

Vol 82 (11) ◽

pp. 1276-1280 ◽

Cited By ~ 16

Author(s):

Olga V. Nikolaeva ◽

Alexander V. Karasev ◽

Stephen M. Garnsey ◽

Richard F. Lee

Keyword(s):

Citrus Tristeza Virus ◽

Sweet Orange ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa Format ◽

Strong Positive Signal ◽

Tristeza Virus ◽

Stem Pitting ◽

Polyclonal Serum ◽

Important Disease ◽

Citrus Tristeza

Citrus tristeza virus (CTV) complex comprises a number of isolates or strains producing several economically important disease syndromes in commercial Citrus spp. The stem pitting syndrome is the most important, and causes substantial losses in many citrus-producing regions of the world. In an attempt to develop a serological tool to rapidly differentiate stem pitting isolates of CTV, we evaluated many combinations of trapping and detecting antibodies in an indirect double-antibody sandwich (I-DAS) enzyme-linked immunosorbent assay (ELISA). Two combinations of trapping and detecting antibodies were found suitable for differentiating stem pitting isolates in extracts of infected sweet orange plants. One used a polyclonal serum raised against bacterially expressed CTV coat protein (CP) for trapping and a conformational monoclonal antibody 3E10 for detection, and the other used two polyclonal antisera generated against bacterially expressed CTV CP. Seventy-six CTV isolates from 20 countries, including 35 that cause stem pitting in sweet orange plants, were analyzed in I-DAS-ELISA using different combinations of polyclonal and monoclonal antibodies for trapping and as intermediate detecting antibodies. The ELISA format developed produces a strong positive signal for CTV isolates that cause stem pitting in sweet orange plants and a negative ELISA signal for CTV isolates that do not cause stem pitting. When combined with data on a universal ELISA format, i.e., reacting with a broad range of CTV isolates, these selective ELISA formats allowed reliable serological differentiation of CTV isolates that caused stem pitting in infected sweet orange plants.

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Genetic Diversity and Phylogenetic Analysis of Citrus tristeza virus Isolates from Turkey

Advances in Virology ◽

10.1155/2019/7163747 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Gözde Erkiş-Güngör ◽

Bayram Çevik

Keyword(s):

Phylogenetic Analysis ◽

Citrus Tristeza Virus ◽

Molecular Characteristics ◽

Cloning And Sequencing ◽

Phylogenetic Groups ◽

The 1960S ◽

Tristeza Virus ◽

Virus Isolates ◽

Stem Pitting ◽

Citrus Tristeza

The presence of Citrus tristeza virus (CTV) in Turkey has been known since the 1960s and the virus was detected in all citrus growing regions of the country. Even though serological and biological characteristics of CTV have been studied since the 1980s, molecular characteristics of CTV isolates have not been studied to date in Turkey. In this study, molecular characteristics of 15 CTV isolates collected from different citrus growing regions of Turkey were determined by amplification, cloning, and sequencing of their major coat protein (CP) genes. The sequence analysis showed that the CP genes were highly conserved among Turkish isolates. However, isolates from different regions showed more genetic variation than isolates from the same region. Turkish isolates were clustered into three phylogenetic groups showing no association with geographical origins, host, or symptoms induced in indicator plants. Phylogenetic analysis of Turkish isolates with isolates from different citrus growing regions of the world including well-characterized type isolates of previously established strain specific groups revealed that some Turkish isolates were closely related to severe quick decline or stem pitting isolates. The results demonstrated that although CTV isolates from Turkey are considered biologically mild, majority of them contain severe components potentially causing quick decline or stem pitting.

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Stem Pitting Strains of Citrus Tristeza Virus in Indonesia

International Organization of Citrus Virologists Conference Proceedings (1957-2010) ◽

10.5070/c5160069w8 ◽

1991 ◽

Vol 11 (11) ◽

Author(s):

A. Muharam ◽

A. M. Whittle

Keyword(s):

Citrus Tristeza Virus ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Tristeza

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Stem-Pitting Citrus tristeza virus Predominantly Transmitted by the Brown Citrus Aphid from Mixed Infections Containing Non-Stem-Pitting and Stem-Pitting Isolates

Plant Disease ◽

10.1094/pdis-10-10-0772 ◽

2011 ◽

Vol 95 (8) ◽

pp. 913-920 ◽

Cited By ~ 5

Author(s):

R. H. Brlansky ◽

Avijit Roy ◽

V. D. Damsteegt

Keyword(s):

Citrus Tristeza Virus ◽

Fruit Size ◽

Haplotype Diversity ◽

Aphid Transmission ◽

Single Strand Conformation Polymorphism ◽

Mixed Infections ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Spp ◽

Citrus Tristeza

Citrus tristeza virus (CTV) is a phloem-limited Closterovirus that produces a variety of symptoms in various Citrus spp. One of these symptoms is stem pitting (SP). SP does not occur in all Citrus spp. but when it does it may cause low tree vigor, decline, and an economic reduction in fruit size and yield. Historically, the first appearance of CTV-SP in a citrus area often occurs after the introduction of the most efficient CTV vector, the brown citrus aphid (BCA), Toxoptera citricida. Hypotheses for this association range from the introduction of these strains in new planting materials to the increased ability of BCA to transmit SP strains from existing CTV sources. It is known that CTV often exists as a complex of isolates or subisolates. Single and multiple BCA transmissions have been used to separate different genotypes or strains of CTV from mixed CTV infected plants. This study was initiated to determine what the BCA transmits when an exotic severe SP CTV isolate B12 from Brazil or B408 from Dominican Republic are mixed with a non-SP (NSP) isolate, FS627 from Florida. Biological and molecular data was generated from grafted mixtures of these isolates and their aphid-transmitted subisolates. Single-strand conformation polymorphism patterns of the 5′ terminal region of open reading frame (ORF) 1a, the overlapping region of ORF1b and ORF2, and the major coat protein gene region of NSP and SP CTV-grafted plants remained unchanged but the patterns of doubly inoculated plants varied. The haplotype diversity within SP isolates B12, B408, and mixtures of NSP and SP isolates (FS627/B12 and FS627/B408) and aphid-transmitted subisolates from doubly inoculated plants was determined by analysis of the haplotype nucleotide sequences. Aphid transmission experiments, symptoms, and molecular analyses showed that SP-CTV was more frequently transmitted with or without NSP-CTV from mixed infections.

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Breakdown of Cross-Protection of Grapefruit from Decline-Inducing Isolates of Citrus tristeza virus Following Introduction of the Brown Citrus Aphid

Plant Disease ◽

10.1094/pdis.2003.87.9.1116 ◽

2003 ◽

Vol 87 (9) ◽

pp. 1116-1118 ◽

Cited By ~ 8

Author(s):

C. A. Powell ◽

R. R. Pelosi ◽

P. A. Rundell ◽

M. Cohen

Keyword(s):

Immunosorbent Assay ◽

Citrus Tristeza Virus ◽

Cross Protection ◽

Enzyme Linked Immunosorbent Assay ◽

Experimental Area ◽

Toxoptera Citricida ◽

The Cross ◽

Tristeza Virus ◽

Citrus Tristeza

A 21-year-old replicated field planting of 84 ‘Ruby Red’ grapefruit trees cross-protected with three mild isolates of Citrus tristeza virus (CTV) was assessed for decline-inducing and non-decline-inducing isolates of the virus 5 years after the brown citrus aphid (BrCA) (Toxoptera citricida Kirkaldy) first was established in the experimental area. Prior to the introduction of the BrCA, the cross-protecting mild isolates had significantly reduced detectable infection with decline-inducing isolates of CTV for 16 years (average infection of 13% in cross-protected trees compared with 67% in unprotected trees). After the introduction of the BrCA, infections with decline-inducing CTV (measured by enzyme-linked immunosorbent assay) were 57, 81, and 71% for trees protected with three mild isolates, respectively, compared with 95% in unprotected trees. These results suggest that the introduction of BrCA accelerated the breakdown of cross-protection against decline-inducing isolates of CTV in grapefruit.

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Citrus Tristeza Virus Isolates of the Same Genotype Differ in Stem Pitting Severity in Grapefruit

Plant Disease ◽

10.1094/pdis-12-19-2586-re ◽

2020 ◽

Vol 104 (9) ◽

pp. 2362-2368

Author(s):

Glynnis Cook ◽

Beatrix Coetzee ◽

Rachelle Bester ◽

Johannes H. J. Breytenbach ◽

Chanel Steyn ◽

...

Keyword(s):

Citrus Tristeza Virus ◽

Aphid Transmission ◽

Common Source ◽

Citrus Paradisi ◽

Full Genome ◽

Full Genome Sequencing ◽

Reading Frame ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Tristeza

Two isolates of the T68 genotype of citrus tristeza virus (CTV) were derived from a common source, GFMS12, by single aphid transmission. These isolates, named GFMS12-8 and GFMS12-1.3, induced stem pitting with differing severity in ‘Duncan’ grapefruit (Citrus × paradisi [Macfad.]). Full-genome sequencing of these isolates showed only minor nucleotide sequence differences totaling 45 polymorphisms. Numerous nucleotide changes, in relatively close proximity, were detected in the p33 open reading frame (ORF) and the leader protease domains of ORF1a. This is the first report of full-genome characterization of CTV isolates of a single genotype, derived from the same source, but showing differences in pathogenicity. The results demonstrate the development of intragenotype heterogeneity known to occur with single-stranded RNA viruses. Identification of genetic variability between isolates showing different pathogenicity will enable interrogation of specific genome regions for potential stem pitting determinants.

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