Evaluation of microsatellite markers in cultivars of sweet orange (Citrus sinensis [L.] Osbeck)

Sweet orange is considered a very important species in the citrus world market and presents wide morphological variability. However, its characterization at the molecular level by random amplified polymorphic DNA (RAPD) and isozyme markers is not appropriate. Microsatellite or simple sequence repeats (SSRs) have been suggested as ideal for studies in cultures of vegetative propagation and as value markers for mapping in several species. However, information on microsatellite polymorphism in citrus species is scarce. In this work, microsatellite markers (AG-repeats) were developed from an enrichment library of genomic DNA of sweet orange cv. Pera (Citrus sinensis [L.] Osbeck), and 31 cultivars of sweet orange were evaluated. Evaluation of 18 microsatellite primers did not permit differentiation of the varieties studied. New microsatellite primers are being evaluated with the aim of detecting polymorphisms among the cultivars and closely related species to be used in genetic mapping programs.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

LIFE TABLE ANALYSIS OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) INFESTING SWEET ORANGE (CITRUS SINENSIS) IN SAO PAULO

An ecological life table for eggs and nymphs of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) was constructed with data obtained from orange orchards (Citrus sinensis Osbeck) in 2 regions of the State of Sao Paulo, over 4 generations in the period from XI-2006 to V-2007, comprising spring, summer, and fall seasons. Young growing shoots with D. citri eggs present were identified, and live individuals were counted until adult emergence. No predatory arthropods were observed in association with D. citri eggs and nymphs during the study. The mean parasitism of fourth- and fifth-instar nymphs by Tamarixia radiata Waterston (Hymenoptera: Eulophidae) was 2.3%. The durations of the egg-adult period were similar among the 4 generations, ranging from 18.0 to 24.7 d (at mean temperatures ranging from 21.6 to 26.0 degrees C) and followed the temperature requirement models obtained in the laboratory for D. citri. However, survival from the egg to the adult stage for the same period varied considerably from 1.7 to 21.4%; the highest mortalities were observed in the egg and small nymphal (first- to thirdinstar) stages, which were considered to be key phases for population growth of the pest.

Relationship between sweet orange yield and intensity of Citrus Variegated Chlorosis

Citrus Variegated Chlorosis (CVC) is currently present in approximately 40% of citrus plants in Brazil and causes an annual loss of around 120 million US dollars to the Brazilian citrus industry. Despite the fact that CVC has been present in Brazil for over 20 years, a relationship between disease intensity and yield loss has not been established. In order to achieve this, an experiment was carried out in a randomized block design in a 3 x 2 factorial scheme with 10-year-old Natal sweet orange. The following treatments were applied: irrigation with 0, 50 or 100% of the evapotranspiration of the crop, combined with natural infection or artificial inoculation with Xylella fastidiosa, the causal agent of CVC. The experiment was evaluated during three seasons. A negative exponential model was fitted to the relationships between yield versus CVC severity and yield versus Area Under Disease Progress Curve (AUDPC). In addition, the relationship between yield versus CVC severity and canopy volume was fitted by a multivariate exponential model. The use of the AUDPC variable showed practical limitations when compared with the variable CVC severity. The parameter values in the relationship of yieldCVC severity were similar for all treatments unlike in the multivariate model. Consequently, the yieldCVC intensity relationship (with 432 data points) could be described by one single model: y = 114.07 exp(-0.017 x), where y is yield (symptomless fruit weight in kg) and x is disease severity (R2 = 0.45; P < 0.01).

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model "diploid embryogenic callus protoplast + diploid mesophyll-derived protoplast". Protoplasts were isolated from embryogenic calli of 'Pera' and 'Westin' sweet orange cultivars (Citrus sinensis) and from young leaves of 'Fremont', Nules', and 'Thomas' mandarins (C. reticulata), and 'Nova' tangelo [C. reticulata x (C. paradisi x C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when 'Pera' sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of 'Pera' sweet orange + 'Fremont' mandarin, 3 'Pera' sweet orange + 'Nules' mandarin, and 2 'Pera' sweet orange + 'Nova' tangelo, and all the diploid regenerated plants showed the 'Pera' sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96 h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.

Molecular characterization of Citrus tristeza virus isolates from Cyprus on the basis of the coat protein gene

Within the context of a program in Cyprus for the control of Citrus tristeza virus (CTV), the coat protein (CP) genes of 12 local isolates of the virus that induced different symptoms on host trees, were compared to those of known isolates. The CP genes were reverse-transcribed (RT) and amplified by polymerase chain reaction (PCR) and the resulting amplicons were cloned and sequenced. Nucleotide sequence analysis revealed no signs of geographic speciation. All the sequences obtained clustered close to those of previously known isolates of worldwide origin that are in five distinct groups. The nucleotide diversity was high compared to that found using a worldwide database of CP gene sequences. These data support the existence of different CTV introductions into Cyprus or an introduction from a location in which CTV is relatively diverse. Some of the isolates induced stem pitting on branches of grapefruit and sweet orange. Such isolates have not been noted often in the Mediterranean basin. They were close in CP sequence to isolate B249 from Venezuela, which induces stem pitting, and are of particular concern for the whole region.

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.

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 rootstock 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. Downregulation 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.

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

Spatial distribution of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in citrus orchards

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Geostatistical characterization of the spatial distribution of Xylella fastidiosa sharpshooter vectors on citrus

A distribuição espacial das espécies de cigarrinhas (Dilobopterus costalimai Young, Acrogonia sp. e Oncometopia facialis Signoret), vetoras da Xylella fastidiosa, agente causal da Clorose Variegada dos Citros, foi estudada com o uso da geoestatística. As avaliações foram feitas em um pomar comercial de laranja 'Pêra' (Citrus sinensis [L.] Osb.), objetivando estabelecer meios para melhor controle dos vetores e da doença. O monitoramento da ocorrência das cigarrinhas no pomar foi feito através de amostragens mensais, utilizando-se armadilhas adesivas amarelas de 3 x 5, distribuídas uniformemente em 50 pontos na área, dispostas em laranjeiras à altura de 1,5 m do solo e substituídas mensalmente. Acrogonia sp. foi a espécie prevalente nas amostragens. Os resultados possibilitaram ajustar modelos aos semivariogramas da distribuição espacial das três espécies no pomar estudado. Durante os três anos consecutivos de amostragem, as populações de Acrogonia sp., D. costalimai e O. facialis apresentaram modelos de distribuição agregada somente nos meses de verão, inverno e primavera, respectivamente, mostrando a necessidade de monitoramento constante desses vetores para reduzir a sua população em épocas favoráveis ao seu desenvolvimento. Através de parâmetros geoestatísticos foi possível calcular a área de agregação das cigarrinhas no pomar. A espécie Acrogonia sp. apresentou área média de agregação de 15.760 m², enquanto para O. facialis e D. costalimai foi possível constatar áreas médias de agregação de 11.555 m² e 10.980 m², respectivamente. Esses resultados indicaram que para um levantamento seguro de cigarrinhas é necessário pelo menos dispor de uma armadilha por hectare.

Effect of temperature, leaf wetness, and rainfall on the production of Guignardia citricarpa ascospores and on back spot severity on sweet orange

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of fruits of the 'Valência' and 'Natal' sweet orange varieties to Guignardia citricarpa and the influence of the coexistence of healthy and symptomatic fruits

Em pomar de laranjeiras 'Valência' e 'Natal' avaliou-se a importância da presença de frutos sintomáticos da mancha preta citros (MPC) na severidade da doença nos frutos cítricos da safra subseqüente. Adicionalmente, avaliou-se o estádio de suscetibilidade dos frutos dessas variedades. Frutos foram protegidos com sacos de papel cristal a partir do estádio de 75% de pétalas caídas em outubro de 2000, até abril de 2001. Frutos foram expostos, em intervalos semanais, da 1ª à 24ª semana. Esse processo se deu tanto em plantas onde os frutos da safra remanescente foram previamente colhidos, como naquelas cujos frutos sintomáticos da safra remanescente permaneceram até a sua queda natural. Avaliou-se a severidade da doença usando uma escala de notas que variou de 0 (ausência de sintomas) a 6 (sintomas severos). Observou-se que para as duas variedades os conídios de Phyllosticta citricarpa, formados nas lesões dos frutos da safra remanescente, não provocaram incremento significativo na severidade da doença dos frutos da safra subseqüente. A proteção dos frutos até 10ª semana após a queda de pétalas não influenciou na quantidade final de lesões, indicando que as descargas de ascósporos que ocorreram a partir desse momento foram, provavelmente, responsáveis pela severidade da doença. Frutos que ficaram expostos entre a 20ª a 24ª semanas após a queda de 75% de pétalas mostraram-se sintomáticos, indicando que nesse estádio frutos encontravam-se suscetíveis ao patógeno.

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in 'Valencia' oranges

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transmission of Citrus leprosis virus C by Brevipalpus phoenicis (Geijskes) to Alternative Host Plants Found in Citrus Orchards

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)