ABSTRACT Despite its interesting agricultural traits, Swingle citrumelo (Citrus paradisi Macfad. ‘Duncan’ × Poncirus trifoliata L. Raf.) rootstock exhibits severe incompatibility when grafted with Pera sweet orange [Citrus sinensis (L.) Osb.] scions, and this fact limits its use for propagation purposes. Nonetheless, the existence of certain Pera sweet orange clones that are compatible with Swingle citrumelo represents a substantial gain for the entire citrus industry. This study investigated the compatibility between seven Pera sweet orange clones and Swingle citrumelo rootstock, regarding the presence of a necrotic line at the graft union junction, the difference in diameter between rootstock and scion, the influence of clonal cleaning by micrografting, and the horticultural performance of the selected combinations. No trees died because of grafting incompatibility, even after twenty years (trial I), except for clone CV1, or after six years and nine months (trial II) from planting. The rootstock/scion trunk diameter ratio was not related to the presence of a necrotic line in the graft-union region. Additionally, these variables did not influence fruit yield and quality, and they were not affected by clonal cleaning or pre-immunisation of the old Pera sweet orange clones.

A limpeza clonal seguida de pré-imunização com complexos protetivos do Citrus tristeza virus(CTV) possibilitaram o cultivo comercial da laranja Pêra, variedade de relevante importância para a citricultura brasileira, mas sensível ao vírus. O emprego de isolados fracos protetivos em outras variedades de citros, mesmo com maior tolerância ao CTV, pode ser também de interesse para evitar a dispersão de isolados severos. O objetivo desta pesquisa foi caracterizar, por meio da análise SSCP (Single Strand Conformational Polymorphism) do gene da proteína do capsídeo, isolados de CTV presentes em plantas das cultivares de laranjas doce Pêra, Hamlin e Valência, propagadas de quatro fontes de borbulhas: 1) clones velhos, 2) clones nucelares, 3) clones microenxertados e 4) clones microenxertados pré-imunizados com o isolado fraco protetivo 'PIAC' do CTV. Avaliou-se ainda a correlação dos padrões SSCP obtidos com intensidade de caneluras no lenho, vigor e produção de frutos. Resultados do SSCP demonstraram baixa diversidade genética entre isolados presentes em diferentes plantas da mesma variedade e mesma fonte de borbulhas, e em alguns casos, em diferentes fontes de borbulhas e variedades. Para os sintomas de tristeza, observou-se menores intensidades em plantas dos clones novos, microenxertados e, dos microenxertados pré-imunizados sobre clones velhos das três variedades. Os padrões SSCP e a sintomatologia observados sugeriram que complexos mais fortes do CTV estão infectando as plantas dos clones velhos das três variedades. A estabilidade do complexo protetivo foi observada nos padrões SSCP dos isolados de CTV de alguns dos clones microenxertados e pré-imunizados. Concluiu-se que as alterações detectadas em outros perfis eletroforéticos deste tratamento não provocaram a perda da capacidade protetiva do isolado 'PIAC' de CTV, inoculado na pré-imunização.

Clonal cleaning, followed by pre-immunization with protective complexes of Citrus tristeza virus(CTV), allowed the commercial cultivation of Pêra sweet orange, a variety that has great importance for Brazilian citriculture but is sensitive to the virus. The use of mild protective isolates in other citrus varieties, even those more tolerant to CTV, can also be of interest to prevent the spread of severe isolates. The aim of this study was to characterize, by means of SSCP (Single Strand Conformational Polymorphism) analysis of the coat protein gene, CTV isolates present in plants of the sweet orange cultivars Pêra, Hamlin and Valencia propagated from four budwood sources: 1) old lines, 2) nucellar lines, 3) shoot-tip-grafted lines, and 4) shoot-tip-grafted lines pre-immunized with the mild CTV protective isolate 'PIAC'. We also evaluated the correlation of the obtained SSCP patterns to stem pitting intensity, tree vigor and fruit yield. SSCP results showed low genetic diversity among the isolates present in different trees of the same variety and same budwood source and, in some cases, in different budwood sources and varieties. Considering tristeza symptoms, lower intensity was noted for plants of new, shoot-tip-grafted and pre-immunized shoot-tip-grafted lines, compared to old lines of the three varieties. The observed SSCP patterns and symptomatology suggested that more severe CTV complexes infect the plants of old lines of all three varieties. The protective complex stability was observed in the SSCP patterns of CTV isolates of some shoot-tip-grafted and pre-immunized clones. It was concluded that the changes detected in other electrophoretic profiles of this treatment did not cause loss of the protective capacity of CTV isolate 'PIAC' inoculated in the pre-immunization.

The use of tolerant rootstocks and preimmunization has satisfactorily controlled losses associated with the Citrus tristeza virus (CTV). Several researchers have shown that CTV mild isolates that are selected in the same region where they are used are superior to isolates obtained from other areas. Thus, budwoods of 20 outstanding citrus trees were collected in north and northwestern Paraná state (Brazil) citrus-producing areas and established to be used in a preimmunization program. These budwoods were tested to evaluate the potential protection of the inherently present viral complex. Based on biological indexing and molecular characterization of the capsid protein gene by RFLP (restriction fragment length polymorphism, which indicated that the plants were infected with mild isolates of CTV), some of the selected plants could be used in a preimmunization program. These potentially mild and protective isolates were challenged with severe 'Rolândia' isolate inoculations by grafting and by the brown citrus aphid (Toxoptera citricida Kirkaldy) vector, which was faster in transmitting the virus. Some isolates had a better protective value than others, particularly when challenged with the severe CTV isolate. The SSCP (single strand conformational polymorphism) molecular analysis was an excellent complementary tool for monitoring the performance of the experiments and the stability of the viral complex present in the plants. Isolate number 1, collected in the municipality of Cruzeiro do Sul (CS-1), was the most promising for protecting commercial Pêra sweet orange (C. sinensis L.) orchards in northern and northwestern Paraná. The Rolândia severe CTV isolate was stable and had a high genetic divergence among the severe isolates used as a control (Capão Bonito and Barão B) and all of the isolates tested.

Plants of Pera sweet orange on Rangpur lime rootstocks, from orchards of the northwest and north of Paraná state, Brazil, were evaluated for severity of symptoms and genetic diversity of Citrus tristeza virus. The severity of symptoms was evaluated by the development of tree, fruit size and stem pitting symptoms. Isolates that infect these plants were compared with known mild and severe isolates by analysis of restriction fragments length polymorphism (RFLP) of the coat protein nucleotide sequences (CPNS), amplified by the polymerase chain reaction (PCR) and undergone digestion with the restriction enzymes Hinf I and Rsa I. The severity of symptoms showed that the analyzed plants from the northwest orchards presented mild to moderate tristeza symptoms, while the plants from the north orchards exhibited moderate to severe symptoms. The RFLP analysis revealed that the CTV isolates are constituted by haplotype mixtures. Rsa I was the enzyme that best discriminated the genetic diversity among the analyzed isolates of CTV. Two main groups were generated by the UPGMA analysis. The isolates from the northwest orchards grouped with most of the mild isolates used as control, and a great part of the isolates from the north orchards, was correlated with the severe isolate Capão Bonito. Correlation between the stem pitting intensity and RFLP patterns, was demonstrated with some exceptions. The failure of protection of some isolates and the contamination of the rootstocks by the severe isolates, in field nursery conditions, before grafting with scions with mild isolates, were the hypotheses considered to explain the occurrence of severe CTV isolates in the North area of Paraná State.

O objetivo deste trabalho foi monitorar a manutenção da estabilidade de isolados protetores contra Citrus tristeza virus (CTV) em clones selecionados de laranja 'Pêra' (Citrus sinensis) pré-imunizados ou infectados naturalmente pelo vírus, após sucessivas propagações clonais. O trabalho foi realizado em condições de campo, no norte do Estado do Paraná. A análise do gene da capa protéica (GPC) de 33 isolados, coletados de 16 clones de laranjeira 'Pêra', foi realizada com o uso da técnica polimorfismo conformacional da fita simples (SSCP). Inicialmente, os isolados foram caracterizados por meio de sintomas de caneluras observados nos clones. Em seguida, o genoma viral foi extraído e utilizado como molde para a amplificação do GCP com uso da transcrição reversa da reação em cadeia da polimerase (RTPCR). Os perfis eletroforéticos dos produtos da RTPCR foram analisados com emprego do coeficiente de Jaccard e do método UPGMA. A maioria dos clones apresentou sintomas fracos a moderados de caneluras, bem como alterações nos perfis dos isolados de CTV. Contudo, a estabilidade dos complexos protetores foi mantida, com exceção dos isolados presentes em dois dos clones analisados. Foi observada baixa variabilidade genética nos isolados durante os anos avaliados.

The objective of this work was to monitor the maintenance of Citrus tristeza virus (CTV) protective isolates stability in selected clones of 'Pêra' sweet orange (Citrus sinensis), preimmunized or naturally infected by the virus, after successive clonal propagations. The work was carried out in field conditions in the north of Paraná State, Brazil. Coat protein gene (CPG) analysis of 33 isolates collected from 16 clones of 'Pêra' sweet orange was performed using single strand conformational polymorphism (SSCP). Initially, the isolates were characterized by symptoms of stem pitting observed in clones. Then viral genome was extracted and used as template for the amplification of CPG by reverse transcription polimerase chain reaction (RTPCR). RTPCR products electrophoretic profiles were analyzed using the Jaccard coefficient and the UPGMA method. The majority of the clones had weak to moderate stem pitting symptoms and its CTV isolates showed alterations in the SSCP profiles. However, the stability of the protective complex has been maintained, except for isolates from two analised clones. Low genetic variability was observed within the isolates during the studied years.

O experimento teve como objetivo caracterizar biológica e molecularmente os isolados de CTV, obtidos de árvores de laranja Pêra em pomares das regiões Norte e Noroeste do Paraná, visando selecionar isolados fracos do Citrus tristeza virus com capacidade protetiva para pré-imunização. Os resultados apontaram sintomas fracos a moderados de caneluras e revelaram similaridade genética da maioria dos isolados analisados e o isolado fraco controle, sugerindo que as plantas selecionadas estão infectadas por haplótipos fracos de CTV, com potencial para serem utilizadas como plantas matrizes.

A cross-protection program citrus tristeza disease, caused by Citrus tristeza virus (CTV) has been developed in the Parana State, to obtain Pera orange mother plants. Thus, 20 plants were selected in the orange commercial fields of the North and Northwest region. Comparisons between mild and severe recognized isolates were made. The results showed mild-to-moderate stem pitting symptoms and the great majority of the isolates grouped up with the mild control isolate. These results suggest that the plants selected were infected by CTV mild haplotypes showing that they have mother plant potential.

Isolados do vírus da tristeza dos citros (CTV) de 35 acessos de pomelos que fazem parte do Banco Ativo de Germoplasma de Citros, localizado no Centro APTA Citros Sylvio Moreira, Cordeirópolis, São Paulo, Brasil, pertencente ao Instituto Agronômico de Campinas (IAC), foram caracterizados através dos sintomas observados nas árvores, indexação biológica, diagnóstico imunológico e análise SSCP (polimorfismo de conformação de fita simples) do gene da proteína do capsídeo. O grupo de plantas que, em geral, apresentou menor volume de copa e severo sintoma de canelura diferenciou-se significativamente do grupo com maior desenvolvimento vegetativo e fraco a moderado sintoma de canelura. No entanto, a maioria dos isolados de CTV das plantas de ambos os grupos induziu fraca reação em limão galego e nenhuma relação entre títulos do vírus nos tecidos e sintomatologia foi observada na avaliação sorológica conduzida por DAS-ELISA. A reação com diferentes anticorpos monoclonais e os distintos padrões eletroforéticos obtidos por SSCP demonstraram que há uma grande diversidade entre os isolados de CTV que infectam os acessos de pomelos. Alta complexidade de bandas dentro de um mesmo isolado foi também observada nos perfis SSCP, demonstrando que cada isolado é constituído por uma mistura de diferentes haplótipos de CTV. Padrões SSCP semelhantes foram observados entre algumas plantas com fortes sintomas de caneluras e entre algumas plantas com sintomas fracos ou moderados de caneluras.

Citrus tristeza virus (CTV) isolates from 35 grapefruit accessions belonging to Citrus Active Germplasm Bank of the "Instituto Agronômico de Campinas" located at the "Centro APTA Citros Sylvio Moreira", Cordeirópolis, São Paulo state, Brazil, were characterized and evaluated through symptoms in the trees, biological indexing, immunological diagnosis with different monoclonal antibodies and SSCP analysis (single-strand conformation polymorphism) of the coat protein gene. Symptomatology indicated that, in general, the group of plants with smaller canopy volume and severe stem pitting differed significantly from the group that presented greater vegetative development and mild to moderate stem pitting. However, the isolates from most of the accessions induced mild reaction on Mexican lime. The serological evaluation through the DAS-ELISA using monoclonal antibodies did not reveal any association between virus titer in the plant tissue and symptoms. The reaction with different monoclonal antibodies and the distinct electrophoresis patterns obtained through SSCP showed that there is a high degree of diversity among the isolates that infect these grapefruit accessions. High complexity within the same isolate was also observed in the SSCP profiles. This finding indicates that the CTV isolates from these plants are a complex mixture of CTV haplotypes. Similar SSCP banding patterns were observed among some plants with strong stem pitting symptoms, and among some plants with weak or moderate stem pitting symptoms.

Estudou-se, mediante polimorfismo enzimático em gel de poliacrilamida, a variabilidade genética das espécies de laranja-doce (Citrus sinensis); laranja-azeda (C. aurantium); tangerinas clementina (C. clementina), sunki (C. sunki), cleópatra (C. reshni) e poncã (C. rsticulata); lima-da-pérsia (C. limettioides); limão-galego (C. aurantifolia); limão-cravo (C. limonia) e trifoliata (Poncirus trifoliata). Extratos de folhas foram analisados para as isoenzimas de malato deidrogenase (MDH), enzima málica (ME), leucino amino peptidase (LAP), glutamato oxaloacetato transaminase (GOT), fosfoglucoisomerase (PGI), fosfoglucomutase (PGM) e isocitrato deidrogenase (IDH). Verificou-se grande variabilidade genética interespecífica, porém nenhuma entre os cultivares de laranja-doce. Foram encontradas algumas aloenzimas, além das referidas pela literatura em gel de amido, como aquelas de uma região próxima ao loco conhecido por Pgm-1, responsável por proteínas monoméricas. Este sistema, denominado PGM, revelou a maior diferenciação entre as espécies, tendo apresentado duas regiões distintas com 9 alelos. No sistema MDH, foram considerados dois locas codificando para proteínas diméricas com 7 alelos; no ME, um loco com 3 alelos; no LAP, possivelmente dois locos responsáveis por proteínas monoméricas com 4 alelos; no GOT, dois focos com 7 alelos; no PGI, um loco com 3 alelos e no IDH, um loco com 4 alelos.

The genetic diversity of citrus cultivars was studied by polyacrylamide gel electrophoresis on sweet orange (C. sinensis); tangerines (C. clementine, C. sunki, C. reshni, C. reticulata); Palestine lime (C. Iimettioides); West Indian lime (C. aurantifolia); Rangpur lime (C. limonia), Sour orange (C. aurantium) and Poncirus trifoliata. Citrus leaf extracts were analysed for isozymes of malato dehidrogenase (MDH), malic enzyme (ME), leucine aminopeptidase (LAP), glutamate oxaloacetate transaminase (GOT), phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM) and isocitrate dehydrogenase (IDH). Interespecific differences were observed; but, none between intraespecific C. sinensis cultivars. Some allozymes were observed in addition to those reffered in the literature on starch gel; e.g., allozymes located next to the known Pgm-1 loci. The PGM system revealed the best differentiation between the cultivars. It showed monomeric proteins and 9 alleles. The MDH system had two loci with 7 alleles; the ME system one locus with 3 alleles; the LAP system one possible locus next to the known Lap-1 and 4 alleles; the GOT two loci for dimeric proteins with 7 alleles; the PGI one locus with 3 alleles; and, the IDH one locus with 4 alleles.