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Journal of Virology

Walter Maccheroni, Marcos C. Alegria, Christian C. Greggio, João Paulo Piazza, Rachel F. Kamla, Paula R. A. Zacharias, Elliot W. Kitajima, Laura C. Assumpção, Giovana Camarotte, Jussara Cardozo, Elaine C. Casagrande, Fernanda Ferrari, Sulamita F. Franco, Poliana F. Giachetto, Alessandra Girasol, Hamilton Jordão Jr., Vitor H. A. Silva, Leonardo C. A. Souza, Carlos I. Aguilar-Vildoso, Almir S. Zanca, Paulo Arruda, João Paulo Kitajima, Fernando C. Reinach, Jesus A. Ferro, Ana C. R. da Silva

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

The outbreak of new diseases in citrus culture has always challenged the producers and researchers urging the development of new strategies for identification of the causative agents and disease control. Several citrus diseases have been related to virus infection (13). They caused severe economical losses for the world citrus industry in the past and are still a problem. Among these viruses the most important is Citrus tristeza virus (CTV), a member of the family Closteroviridae, which induces diseases such as quick decline, causing the death of trees grafted on sour orange (Citrus aurantium) rootstock and stem pitting of scion cultivars regardless of the rootstock type (5).

Citrus Tristeza was first detected in Brazil in later 1930s, killing millions of sweet orange (C. sinensis) trees grafted on sour orange. The problem was solved by exchanging the sensitive sour orange with a tolerant Rangpur lime (C. limonia) rootstock. Today, more than 85% of the 200 million trees in the country are grafted on Rangpur lime (18).

In 1999, a new citrus disease was identified in the state of Minas Gerais, Brazil, and was named citrus sudden death (CSD). The disease is mainly characterized by a rapid decline of scions grafted on Rangpur lime or Volkamer lemon (C. volkameriana). Rootstocks such as Cleopatra mandarin (C. reshni), Sunki mandarin (C. sunki), and trifoliates (Poncirus trifoliata) and their hybrids (citranges and citrumelos) are apparently tolerant to the causal agent, and these rootstocks are presently being used as alternatives to Rangpur lime and for inarching purposes as a measure to recover CSD-affected trees (630). The number of dead trees affected by the disease dramatically increased from 500 trees in 1999 to at least 1 million in 2003. This number would be even greater if we considered those infected but asymptomatic. The disease has progressed rapidly from the original focus in the southwest of Minas Gerais state towards the northern part of São Paulo state (30).

Symptomatic plants present a pale green coloration of the whole canopy, different levels of defoliation, the absence of new shoots, and death of the root system. General decline symptoms and plant death has been related to rootstock phloem degeneration in the bud union region (30). CSD is also characterized by the development of a strong yellow stain in the phloem of the Rangpur lime and Volkamer lemon rootstocks. The plant physiological status is important in disease progression, since the severity of the symptoms increases under conditions of high water demand. Death of infected trees occurs between 1 and 12 months after the appearance of symptoms, depending on the season and citrus variety. The disease has a period of incubation of at least 2 years before symptoms are detected (186).

The spatial and temporal patterns of CSD dissemination has been remarkably similar to those seen with the spread of CTV, which is mainly transmitted by the aphid Toxoptera citricida in Brazil (6). The similarities between the symptoms of CSD and CTV decline lead researchers to suggest that the new disease is probably caused by a new strain of CTV. Alternatively, a different virus could be the causative agent of the CSD disease (630). Several attempts to associate CSD with a CTV mutant have failed (1435).

Combining massive shotgun sequencing and bioinformatic tools, we were able to identify a new virus present only in plants from the affected area and strongly associated with CSD symptoms.

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Identification and genomic characterization of a new virus (Tymoviridae family) associated with Citrus sudden death disease
79

Walter Maccheroni, Marcos C. Alegria, Christian C. Greggio, João Paulo Piazza, Rachel F. Kamla, Paula R. A. Zacharias, Elliot W. Kitajima, Laura C. Assumpção, Giovana Camarotte, Jussara Cardozo, Elaine C. Casagrande, Fernanda Ferrari, Sulamita F. Franco, Poliana F. Giachetto, Alessandra Girasol, Hamilton Jordão Jr., Vitor H. A. Silva, Leonardo C. A. Souza, Carlos I. Aguilar-Vildoso, Almir S. Zanca, Paulo Arruda, João Paulo Kitajima, Fernando C. Reinach, Jesus A. Ferro, Ana C. R. da Silva

Identification and genomic characterization of a new virus (Tymoviridae family) associated with Citrus sudden death disease

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

The outbreak of new diseases in citrus culture has always challenged the producers and researchers urging the development of new strategies for identification of the causative agents and disease control. Several citrus diseases have been related to virus infection (13). They caused severe economical losses for the world citrus industry in the past and are still a problem. Among these viruses the most important is Citrus tristeza virus (CTV), a member of the family Closteroviridae, which induces diseases such as quick decline, causing the death of trees grafted on sour orange (Citrus aurantium) rootstock and stem pitting of scion cultivars regardless of the rootstock type (5).

Citrus Tristeza was first detected in Brazil in later 1930s, killing millions of sweet orange (C. sinensis) trees grafted on sour orange. The problem was solved by exchanging the sensitive sour orange with a tolerant Rangpur lime (C. limonia) rootstock. Today, more than 85% of the 200 million trees in the country are grafted on Rangpur lime (18).

In 1999, a new citrus disease was identified in the state of Minas Gerais, Brazil, and was named citrus sudden death (CSD). The disease is mainly characterized by a rapid decline of scions grafted on Rangpur lime or Volkamer lemon (C. volkameriana). Rootstocks such as Cleopatra mandarin (C. reshni), Sunki mandarin (C. sunki), and trifoliates (Poncirus trifoliata) and their hybrids (citranges and citrumelos) are apparently tolerant to the causal agent, and these rootstocks are presently being used as alternatives to Rangpur lime and for inarching purposes as a measure to recover CSD-affected trees (630). The number of dead trees affected by the disease dramatically increased from 500 trees in 1999 to at least 1 million in 2003. This number would be even greater if we considered those infected but asymptomatic. The disease has progressed rapidly from the original focus in the southwest of Minas Gerais state towards the northern part of São Paulo state (30).

Symptomatic plants present a pale green coloration of the whole canopy, different levels of defoliation, the absence of new shoots, and death of the root system. General decline symptoms and plant death has been related to rootstock phloem degeneration in the bud union region (30). CSD is also characterized by the development of a strong yellow stain in the phloem of the Rangpur lime and Volkamer lemon rootstocks. The plant physiological status is important in disease progression, since the severity of the symptoms increases under conditions of high water demand. Death of infected trees occurs between 1 and 12 months after the appearance of symptoms, depending on the season and citrus variety. The disease has a period of incubation of at least 2 years before symptoms are detected (186).

The spatial and temporal patterns of CSD dissemination has been remarkably similar to those seen with the spread of CTV, which is mainly transmitted by the aphid Toxoptera citricida in Brazil (6). The similarities between the symptoms of CSD and CTV decline lead researchers to suggest that the new disease is probably caused by a new strain of CTV. Alternatively, a different virus could be the causative agent of the CSD disease (630). Several attempts to associate CSD with a CTV mutant have failed (1435).

Combining massive shotgun sequencing and bioinformatic tools, we were able to identify a new virus present only in plants from the affected area and strongly associated with CSD symptoms.

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