GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

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.

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

Aims: Detection of Xylella fastidiosa in citrus plants and insect vectors.Methods and Results: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay.Conclusions: the use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively.Significance and Impact of the study: the employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.

An experimental inoculation system to study citrus-Xylella fastidiosa interactions

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in expertmental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive Study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stein absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of I-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal), In foam trays, the seedlings grew less, the incubation period was shorter. and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Evaluation of the genetic diversity of Xylella fastidiosa strains from citrus and coffee hosts by single-nucleotide polymorphism markers

The aim of this study was to obtain information about genetic diversity and make some inferences about the relationship of 27 strains of Xylella fastidiosa from different hosts and distinct geographical areas. Single-nucleotide polymorphism (SNP) molecular markers were identified in DNA sequences from 16 distinct regions of the genome of 24 strains of X. fastidiosa from coffee and citrus plants. Among the Brazilian strains, coffee-dependent strains have a greater number of SNPs (10 to 24 SNPs) than the citrus-based strains (2 to 12 SNPs); all the strains were compared with the sequenced strain 9a5c. The identified SNP markers were able to distinguish, for the first time, strains from citrus plants and coffee and showed that strains from coffee present higher genetic diversity than the others. These markers also have proven to be efficient for discriminating strains from the same host obtained from different geographic regions. X. fastidiosa, the causal agent of citrus variegated chlorosis, possesses genetic diversity, and the SNP markers were highly efficient for discriminating genetically close organisms.

An evaluation of the genetic diversity of Xylella fastidiosa isolated from diseased citrus and coffee in São Paulo, Brazil

Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Cafe, respectively, were indistinguish able based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Coffee leaf scorch caused by a strain of Xylella fastidiosa from citrus

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var, robusta 'Apuatao 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosn and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X, fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

Citrus and coffee strains of Xylella fastidiosa induce Pierce's disease in grapevine

Xylella fastidiosa causes citrus variegated chlorosis (CVC) disease in Brazil and Pierce's disease of grapevines in the United States. Both of these diseases cause significant production problems in the respective industries. The recent establishment of the glassy-winged sharpshooter in California has radically increased the threat posed by Pierces disease to California viticulture. Populations of this insect reach very high levels in citrus groves in California and move from the orchards into the vineyards, where they acquire inoculum and spread Pierce's disease in the vineyards. Here we show that strains of X. fastidiosa isolated from diseased citrus and coffee in Brazil can incite symptoms of Pierce's disease after mechanical inoculation into seven commercial Vitis vinifera varieties grown in Brazil and California. Thus, any future introduction of the CVC strains of X. fastidiosa into the United States would pose a threat to both the sweet orange and grapevine industries. Previous work has clearly shown that the strains of X. fastidiosa isolated from Pierce's disease- and CVC-affected plants are the most distantly related of all strains in the diverse taxon X. fastidiosa. The ability of citrus strains of X. fastidiosa to incite disease in grapevine is therefore surprising and creates an experimental system with which to dissect mechanisms used by X.,fastidiosa in plant colonization and disease development using the full genome sequence data that has recently become available for both the citrus and grapevine strains of this pathogen.

Expressão diferencial de genes em laranja doce (Citrus sinensis L. Osb) em tangerina (Citrus reticulata blanco) em resposta à infecção por Xylella fastidiosa

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

Estrutura de populações e inoculações recíprocas de Xylella fastidiosa subsp. pauca com ocorrência em cultivos vizinhos de Citrus sinensis e Coffea arabica sob condições do estado de São Paulo

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Effect of contact and systemic insecticides on the sharpshooter Bucephalogonia xanthophis (Hemiptera: cicadellidae), a vector of Xylella fastidiosa in citrus

The knockdown and toxic effects of insecticides of different chemical groups and modes of action registered for citrus in Brazil were investigated for effective control of Bucephalogonia xanthophis, a sharpshooter vector of Xylella fastidiosa in citrus. The active ingredients dimethoate (1.2 mL/1.2L), imidacloprid (0.24 mL/1.2L) and lambda-cyhalothrin (0.24 mL/1.2L), as well as a control (water), were sprayed onto branches of potted-citrus nursery trees to evaluate the effect of residual contact. The insects were confined on sprayed branches by using sleeve cages, in groups of 10 per branch (5 branches/treatment). Lambdacyhalothrin showed a knockdown effect on B. xanthophis (>70% mortality within 2 h of exposure), and the residues were effective for approximately one wk. Imidacloprid, lambdacyhalothrin and dimethoate suppressed the vector populations for up to 3 wk after application, when the insects were exposed to sprayed plants for at least 24 h. In another experiment, 2 neonicotinoid insecticides (thiamethoxam and imidacloprid) were applied by soil drench to potted nursery trees, in order to study their systemic effect, i.e., mortality by ingestion on sharpshooter adults. Thiamethoxam and imidacloprid effectively controlled the vectors at all concentrations tested, when the insects were exposed to treated plants for 24 h (>80% mortality) or 48 h (near 100% mortality). The knockdown effect of thiamethoxam and lambda-cyhalothrin might be particularly important to prevent vector transmission of X. fastidiosa in citrus groves.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the sigma(54) regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma(54)-dependent manner. A more complete picture of the sigma(54) regulon was achieved by combining the transcriptome data with an in silico search for potential sigma(54)-dependent promoters, using a position weight matrix approach. One of these sigma(54)-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma(54)-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma(54) regulon.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of Sao Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were reisolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Tree performance and fruit yield and quality of `Okitsu` Satsuma mandarin grafted on 12 rootstocks

The citriculture in Brazil, as well as in other important regions in the world, is based on very few mandarin cultivars. This fact leads to a short harvest period and higher prices for off-season fruit. The `Okitsu` Satsuma (Citrus unshiu Marc.) is among the earliest ripening mandarin cultivars and it is considered to be tolerant to, citrus canker (Xanthomonas citri subsp. citri Schaad et al.) and to citrus variegated chlorosis (Xylella fastidiosa Wells et al.). Despite having regular fruit quality under hot climate conditions, the early fruit maturation and absence of seeds of `Okitsu` fruits are well suited for the local market in the summer(December through March), when the availability of citrus fruits for fresh consumption is limited. Yet, only a few studies have been conducted in Brazil on rootstocks for `Okitsu`. Consequently, a field trial was carried out in Bebeclouro, Sao Paulo State, to evaluate the horticultural performance of `Okitsu` Satsuma mandarin budded onto 12 rootstocks: the citrandarin `Changsha` mandarin (Citrus reticulata Blanco) x Poncirus trifoliata `English Small`: the hybrid Rangpur lime (Citrus limonia Osbeck) x `Swingle` citrumelo (P. trifoliata (L.) Raf. x Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L) Raf)`Rubidoux`,`FCAV` and `Flying Dragon`(P. trifoliata var. monstrosa); the mandarins `Sun Chu Sha Kat`(C. reticulata Blanco) and `Sunki`(Citrus sunki (Hayata) Hort. ex. Tanaka); the Rangpur limes (C. limonia Osbeck) `Cravo Limeira` and `Cravo FCAV`;`Carrizo` citrange (Citrus sinensis x P. trifoliata), `Swingle` citrumelo (P. trifoliata x C. paradisi), and `Orlando` tangelo (C. paradisi x Citrus tangerina cv. `Dancy`). The experimental grove was planted in 2001, using a 6 m x 3 m spacing, in a randomized block design. No supplementary irrigation was applied. Fruit yield, canopy volume, and fruit quality were assessed for each rootstock. A cluster multivariate analysis identified three different rootstock pairs with similar effects on plant growth, yield and fruit quality of `Okitsu` mandarin. The `Flying Dragon `trifoliate had a unique effect over the `Okitsu` trees performance, inducing lower canopy volume and higher yield efficiency and fruit quality, and might be suitable for high-density plantings. The `Cravo Limeira` and `Cravo FCAV` Rangpur limes induced early-ripening of fruits, with low fruit quality. `Sun Chu Sha Kat` and `Sunki` mandarins and the `Orlando` tangelo conferred lower yield efficiency and less content of soluble solids for the latter rootstock. (C) 2009 Elsevier B.V. All rights reserved.

Horticultural performance of `Folha Murcha` sweet orange onto twelve rootstocks

Despite its outstanding position, the Brazilian citriculture is established on a very limited pool of varieties that limits its expansion and restricts the fruit availability throughout the year. This situation determines the urgent necessity of developing alternative scion and rootstock cultivars, with good performance under local conditions. `Folha Murcha` sweet orange (Citrus sinensis (L.) Osbeck) is a late-harvest cultivar, suitable both for the juice processing industry and the fresh fruit market, being described as tolerant to citrus canker (Xanthomonas citri subsp. citri Schaad et al.), and less affected by citrus variegated chlorosis (Xylella fastidiosa Wells et al.). A study was conducted in Bebedouro, Sao Paulo State, Brazil, to evaluate the horticultural performance of `Folha Murcha` sweet orange budded onto 12 rootstocks: the citrandarin `Changsha` mandarin (Citrus reticulata Blanco) x Poncirus trifoliata `English Small`: the hybrid `Rangpur` lime (Citrus limonia Osbeck) x `Swingle` citrumelo (P. trifoliata (L.) Raf x Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L.) Raf.)`Rubidoux`, `FCAV`, and `Flying Dragon` (P. trifoliata var. monstrosa); the `Sun Chu Sha Kat` mandarin (C. reticulata Blanco); the `Sunki` mandarin (Citrus sunki (Hayata) Hart. ex. Tanaka); the `Rangpur` limes (C. limonia Osbeck) `Cravo Limeira` and `Cravo FCAV`; `Carrizo` citrange (C. sinensis x P. trifoliata), `Swingle` citrumelo (P. trifoliata x C. paradisi), and `Orlando` tangelo (C. paradisi x Citrus tangerina cv. `Dancy`). The experimental grove was planted in 2001, using a 7 m x 4 m spacing, in a randomized block design, with five replications and two plants per plot. No supplementary irrigation was applied. Fruit yield, canopy volume, tree tolerance to drought and to citrus variegated chlorosis, and fruit quality were assessed for each rootstock. Trees grafted onto the `Flying Dragon` trifoliate were smaller in size, but had largest yield efficiency when compared to those grafted onto other rootstocks. Lower alternate bearing index was observed on trees budded onto `Cravo FCAV` `Rangpur` lime. Both `Rangpur` lime rootstocks and the `Sunki` mandarin induced higher tree tolerance to drought. The `Flying Dragon` trifoliate induced better fruit quality and higher tolerance to citrus variegated chlorosis (CVC) to `Folha Murcha` trees. A cluster multivariate analysis identified three groups of rootstocks with similar effects on `Folha Murcha` tree performance. Among the 12 evaluated rootstocks, the `Flying Dragon` trifoliate has a unique effect on plant growth, tolerance to drought and CVC, fruit yield and fruit quality of `Folha Murcha` trees, and may be better suited for high-density plantings. (C) 2011 Elsevier B.V. All rights reserved.