Dweet Mottle Disease Probably Is Caused by Citrus Leaf Blotch Virus

Sixteenth IOCV Conference, 2005—Other Viruses

Dweet Mottle Disease Probably is Caused by Citrus Leaf Blotch Virus

M. C. Vives, J. A. Pina, J. Juárez, L. Navarro, P. Moreno, and J. Guerri

Instituto Valenciano de Investigaciones Agrarias, 46113 Moncada, Spain

ABSTRACT. Dweet mottle disease was first detected in Cleopatra mandarin CRC-270 in Cali- fornia and causes chlorotic blotching in Dweet tangor seedlings. Nagami kumquat SRA-153 induces similar symptoms in this indicator, but additionally causes vein clearing in several citrus species, stem pitting in Etrog citron, and bud-union crease when propagated on Troyer citrange. Citrus leaf blotch virus (CLBV) was found in this kumquat and its genomic RNA sequenced, thus allowing its detection by molecular hybridization and RT-PCR assays. Two Californian sources of Dweet mottle (DMV-931 and DMV-932) and kumquat SRA-153 were graft-inoculated on Pineap- ple sweet orange, Dweet tangor, Etrog citron and Nules Clementine and infected Clementine buds were then propagated on Carrizo citrange rootstock. Kumquat SRA-153 induced bud-union crease of Nules Clementine on citrange, vein clearing in Pineapple sweet orange, chlorotic blotching in Dweet tangor and stem pitting in Etrog citron, whereas Dweet mottle sources only induced blotching in Dweet tangor and stem pitting in Etrog citron. Fragments of the CLBV genome were RT-PCR amplified from indicator plants inoculated with either kumquat SRA-153 or Dweet mottle sources. The nucleotide identity between CLBV isolate SRA-153 and the DMV-931 and DMV- 932 sources was 96.6% and 96.8% in a region of the RNA replicase gene, and 98.7% and 98.5% in the coat protein gene, respectively. These data and previous findings that Dweet mottle and CLBV are difficult to eliminate by shoot-tip grafting or thermotherapy, suggest that Dweet mottle may be caused by CLBV, and that, besides CLBV, a different pathogen causing bud-union crease and vein clearing may be present in kumquat SRA-153 but not in DMV-931 and DMV-932 sources. Index words. Troyer Citrange, Etrog citron, virus characterization, RT-PCR.

In 1968, routine indexing car- found in Nagami kumquat, clone ried out in the Citrus Variety SRA-153, from Corsica (France). Improvement Program developed in When graft-inoculated to Dweet California, revealed a new pathogen tangor it induced chlorotic blotches in a Cleopatra mandarin tree from resembling those induced by DMV, Florida (CRC 270) that induced but additionally it caused stem pit- chlorotic blotching in Dweet tangor ting in Etrog citron, vein clearing in and a mild exocortis reaction in Pineapple sweet orange and other Etrog citron (8). The parent tree cultivars, and bud union crease showed no visible evidence of dam- when propagated on Troyer citrange age due to any known virus, the rootstock (2, 7). trunk appeared normal and no pit- Citrus leaf blotch virus (CLBV) ting or bark discoloration was evi- has been partially puriﬁed from Na- dent, although the fruits were gami kumquat SRA-153 (3) and its small, the twigs were dying back, genomic RNA (gRNA) completely and little new growth was apparent. sequenced (12). The CLBV gRNA The pathogen responsible for these contains three open reading frames symptoms was graft and mechani- (ORFs) encoding a polyprotein in- cally transmitted to several citrus volved in replication, a potential species and varieties but, since the movement protein and the capsid pathogen caused a reaction only in protein. In addition to the gRNA, in- Dweet tangor, it was named Dweet fected plants contain two sets of sub- mottle virus (DMV) (8). genomic RNAs that are 3’ and 5’ co- In 1984, in routine indexing done terminal, respectively (13). Availabil- at the Citrus Variety Improvement ity of the CLBV sequence allowed the Program developed in Spain, a development of rapid and speciﬁc graft-transmissible pathogen was methods for routine detection of the

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252 Sixteenth IOCV Conference, 2005—Other Viruses virus based on RT-PCR, and dot-blot was then resuspended in 25 µl of and tissue print hybridization proto- DEPC-treated distilled water. Total cols (4, 14). Using these procedures, RNA was used as template to amplify CLBV has been detected in leaf sam- by PCR two genome regions (R and C) ples of different citrus varieties from as previously described (4, 14). Spain, Australia, France, Japan and Region R, located in ORF 1 was USA (3, 4, 14), confirming that this amplified with primers KU-54 (5’- pathogen is widespread in many cit- ACTTGCAGAAATGATCAGACCG-3’, rus growing areas. positions 2260-2281) and KU-55 (5’- In this work we report biological TGCCTCATAGAAATTTATTAATG- and molecular evidence that sug- CAC-3’, positions 2728-2703). Region gests Dweet mottle disease may be C, containing the conserved C-termi- caused by Citrus leaf blotch virus. nal region of the coat protein gene, was amplified with primers KU-18 MATERIALS AND METHODS (5’-TTAAGATTACAGACACGAAGG- 3’, positions 7686-7706) and KU-19 Virus isolates and citrus (5’-CTGTTTTTGAATTTTGCTCG-3’, hosts. The CLBV isolate SRA-153 positions 8123-8104), based on the used in this work was maintained in CLBV sequence (12). plants of Nagami kumquat grafted Cloning, SSCP analysis, on rough lemon. The Californian sequencing, and nucleotide sources of Dweet mottle disease, sequence analysis. The RT-PCR DMV-931 and DMV-932, kindly pro- products were cloned into the vided by D. Gumpf and introduced pGEM-T plasmid vector (Promega) through the citrus quarantine sta- using standard protocols (10), and tion in Moncada, Valencia (Spain), ten randomly selected clones from were on Pineapple sweet orange and each region and isolate were PCR- Satsuma mandarin, respectively. amplified with the corresponding These three inoculum sources primers. The PCR products were were indexed on Pineapple sweet analyzed by SSCP with electro- orange, Dweet tangor, Nules Clem- phoresis in 10% polyacrylamide gels entine, and Etrog citron. Pineapple at 300V for 2.5 h as previously sweet orange and Dweet tangor described (9). The SSCP pattern of plants were seedlings, and Nules individual clones was compared Clementine and Etrog citron were with that of the corresponding RT- propagated on Carrizo citrange and PCR product. The nucleotide rough lemon seedlings, respectively. sequence of the predominant haplo- Two to four bark patches from type for each region and isolate was each virus source were graft inocu- determined with an ABI PRISM lated onto six plants of each indica- DNA sequencer 377 (PE Biosys- tor. Plants were grown in an artificial tems). Sequence alignment was potting mix (50% sand and 50% peat done with the CLUSTAL W program moss) in a temperature-controlled (11) and estimation of the nucleotide (18-26°C) greenhouse and fertilized and amino acidic identities were by a standard procedure (1). done with the MEGA program (6). RNA extraction, reverse tran- scription and polymerase chain RESULTS reaction (RT-PCR) amplification. Total RNA was extracted from Biological indexing in indica- approximately 100 mg of thoroughly tor plants. Six plants of Pineapple trimmed fresh young leaf tissue using sweet orange, Dweet tangor, Etrog TRIzolR reagent (Invitrogen), which citron and Nules Clementine were contains isothyocyanate, following graft inoculated with bark patches the manufacturer’s instructions for of Dweet mottle sources (DMV-931 samples with high sugar content, and and DMV-932), kumquat SRA-153

Sixteenth IOCV Conference, 2005—Other Viruses 253 or healthy kumquat. Six-months-old DMV-931, DMV-932, kumquat SRA- infected Clementine buds were then 153 and healthy kumquat were used propagated on Carrizo citrange as templates with the two selected seedlings (Fig. 1). The first two primer sets. Extracts from DMV- flushes of Pineapple sweet orange 931, DMV-932, and kumquat SRA- and Dweet tangor were observed for 153 sources yielded single RT-PCR leaf symptoms. Etrog citron was products of the expected size for observed for stem pitting 3 to 10 mo region R (469 nt) or for region C after inoculation. Budlings of (438 nt), whereas no amplification infected Nules Clementine on Carr- was obtained using equivalent izo citrange rootstock were exam- extracts from healthy kumquat (Fig. ined 6 and 12 mo after propagation, 2A). CLBV was also detected by RT- by lifting a piece of bark to check for PCR in all symptomatic plants inoc- the presence of bud union crease. ulated with kumquat SRA-153, In all plants tested Kumquat DMV-931, or DMV-932, respectively SRA-153 source induced intense (data not shown). vein clearing in Pineapple sweet Sequence analysis. To charac- orange, intense chlorotic blotching terize the population structure of in Dweet tangor, stem pitting in CLBV in Dweet mottle isolates Etrog citron, and bud union crease DMV-931 and DMV-932, the RT- of Nules Clementine grafted on Car- PCR products from genomic regions rizo citrange, whereas Dweet mot- R and C were analyzed by SSCP. All tle sources DMV-931 and DMV-932 SSCP patterns showed only one or caused only mild chlorotic blotching two intense bands, suggesting that in 3-4 Dweet tangor plants, and each RT-PCR product was most stem pitting in 4-5 Etrog citron likely composed of a single predomi- plants, but no vein clearing or bud nant sequence. To estimate more union crease (Fig. 1). Indicator accurately the population composi- plants budded from healthy kum- tion of these isolates, the RT-PCR quat showed no symptoms. products generated from both Detection of CLBV in Dweet genomic regions were cloned into mottle sources. To determine if pGEM-T vector and ten randomly CLBV was present in Dweet mottle selected clones were PCR amplified sources, total RNA extracts from with the corresponding primers and

Fig. 1. Outline of the inoculations performed and symptoms caused by DMV-931, DMV-932, and SRA-153 inoculum sources.

254 Sixteenth IOCV Conference, 2005—Other Viruses

Fig. 2. RT-PCR detection of CLBV from kumquat SRA-153 and Dweet mottle sources, and SSCP analysis of the genomic regions R (left panels) and C (right panels). An outline of the CLBV genome is at the top. Black boxes indicate the genomic regions analyzed. A) RT-PCR detection of CLBV in total RNA extracts from DMV-931 (lane a), DMV- 932 (lane b), and SRA-153 (lane c) sources, healthy kumquat (lane (-)), and 1-Kb plus DNA ladder (lane M). B) SSCP analysis of 6 cDNA clones (lanes 1-6) of each region from isolate DMV-931. Asterisks indicate the SSCP pattern of the RT-PCR products from which the clones were obtained. C) Nucleotide and amino acid identities (%) between CLBV SRA-153 isolate and Dweet mottle sources DMV-931 and DMV-932. analyzed by SSCP. The SSCP pat- 153 and Dweet mottle sources DMV- terns of these clones were compared 931 and DMV-932, the nucleotide with those of the corresponding RT- sequences of two clones from each PCR products and found to be iden- genomic region and source were tical, indicating that both isolates determined. The comparative nucle- are composed of a predominant otide identities of CLBV isolate sequence variant (Fig. 2B). SRA-153 and DMV-931 and DMV- To estimate the nucleotide and 932 were 96.6 and 96.8% in R amino acidic identities between region, and 98.7 and 98.5% in C CLBV isolates from kumquat SRA- region, respectively, whereas the

Sixteenth IOCV Conference, 2005—Other Viruses 255 amino acid identities with both SRA-153, that also induced blotch- Dweet mottle sources were 99.5% ing in Dweet tangor and stem pit- and 100% in the R and C regions, ting in Etrog citron, but not vein respectively (Fig. 2C). clearing in Pineapple sweet orange or bud union crease on Carrizo cit- DISCUSSION range (2, 3). In a previous study, low genetic The results obtained in this work variation was found between CLBV suggest that CLBV is the causal isolates from different host species agent of Dweet mottle disease. The and geographical origins (15). symptoms induced by CLBV isolate Sequence comparisons between SRA-153 in Dweet tangor and Etrog CLBV found in Dweet mottle citron are indistinguishable from sources and the isolate SRA-153, those caused by both Dweet mottle showed nucleotide identities over sources in the same indicators, and 96.6% and 98.5% and amino acid like CLBV, the pathogen responsible identities of 99.5% and 100% for the for Dweet mottle disease was also R and C regions, respectively, indi- difficult to eliminate by shoot-tip cating that Dweet mottle isolates of grafting (7) or thermotherapy CLBV were closely related to all (unpublished results). Further- other isolates characterized and more, CLBV was detected by RT- confirming genetic stability of PCR in both Dweet mottle sources CLBV. as well as in different indicator In addition to Dweet mottle plants inoculated with them. How- sources, CLBV has been detected in ever, while CLBV SRA-153 induced samples from Corsica (France), bud union crease of Nules clemen- Valencia (Spain), Florida (USA), tine grafted on Carrizo citrange, Japan and Australia, indicating that vein clearing in Pineapple sweet this virus must be widespread (3, 4, orange, chlorotic blotching in Dweet 14). Recently we have reported seed tangor, and stem pitting in Etrog transmission of CLBV in Nagami citron, Dweet mottle sources only kumquat, Carrizo citrange and sour induced chlorotic blotching in Dweet orange (5), a finding that could tangor and stem pitting in Etrog cit- explain in part the dissemination of ron. These differences in symptom this virus throughout the world. To expression suggest that CLBV control CLBV spread during citrus would be responsible only for blotch- propagation is necessary by not only ing in Dweet tangor and stem pit- the use of virus free buds, but also ting in Etrog citron, and symptoms by rootstock seedlings originating of bud union crease and vein clear- from CLBV-free seed sources. ing would be caused by a different pathogen or by an interaction ACKNOWLEDGMENTS between CLBV and other biotic fac- tors. Our results agree with previ- The authors thank M. Boil for ous detection of CLBV in kumquat her excellent technical assistance. lines 38-1 and 497-2, obtained by This work was supported by INIA shoot-tip grafting from kumquat Projects SC93-110 and SC97-103.

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