Ineffectiveness of pruning to control citrus huanglongbing caused by Candidatus Liberibacter americanus

The huanglongbing (HLB) disease of citrus trees, caused by Candidatus Liberibacter asiaticus and Ca. Liberibacter americanus, was first reported in Brazil in March, 2004. The presence of the disease has caused serious concerns among growers. Pruning experiments were conducted to determine if removal of symptomatic branches or the entire canopy (decapitation) would eliminate infected tissues and save HLB-affected trees. Pruning was done in five blocks on a total of 592 3- to 16 year-old 'Valencia', 'Hamlin' or 'Pera' sweet orange trees showing no symptoms or with two levels of symptom severity. Ten decapitated trees per block were caged and all trees were treated with insecticides to control the psyllid vector, Diaphorina citri. Mottled leaves reappeared on most symptomatic (69.2%) as well on some asymptomatic (7.6%) pruned trees, regardless of age, variety, and pruning procedure. Presence of the pathogen (Ca. Liberibacter americanus) in all symptomatic trees was confirmed by PCR. In general, the greater the symptom severity before pruning the lower the percentage of trees that remained asymptomatic after pruning.

Rheological properties of frozen concentrated orange juice (FCOJ) as a function of concentration and subzero temperatures

Data on flow properties of Frozen Concentrated Orange Juice (FCOJ) produced from oranges cv. Pera-Rio (65.04 Brix, 8.8% w/w pulp content, 2.5% w/w pectin, 3.84% citric acid, 1.293 g cm(-3)) from -18 to 0 degrees C were fitted with appropriate predictive models. The power law model was found to be the most appropriate to fit the flow curves obtained for FCOJ between 46.56 and 65.04 degrees Brix. In higher concentrations, thixotropy was observed and showed more temperature dependence. A single equation combining Arrhenius and exponential relationships was applied to describe the temperature effect and shear rate on the quantity of breakdown of FCOJ.

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.

Identification and analysis of single nucleotide polymorphisms (SNPs) in citrus

Most of the cultivated species of citrus have narrow genetic basis. Relationships among species and cultivars are obscured by sexual compatibility, polyembryony, apomixis and a high incidence of somatic mutations. DNA analysis is crucial in genetic studies not only for citrus breeding programs but also for characterization of hybrids and species. In this paper, single nucleotide polymorphisms ( SNPs) were investigated in 58 accessions of Citrus, hybrids and related genera. Genomic sequences of 'Pera IAC' sweet orange ( Citrus sinensis L. Osbeck) were used for primer design and selection of sequence tagged sites (STSs) for identification of SNPs. Analysis of 36 STSs showed identical sequences among 40 of the 41 sweet orange accessions studied. However, these accessions were heterozygous for many SNPs. Ten selected STSs were analyzed in 17 additional accessions from 13 species and hybrids. Comparing to the 'Pera IAC' sweet orange accession, a total of 150 polymorphic nucleotides were identified and most of the alterations were transitions ( 52.7%). The greatest number of SNPs was observed in Poncirus trifoliata ( L.) Raf. and the smallest in 'Ponkan' mandarin ( Citrus reticulata Blanco). At the intra-specific level, 'Bafa Gigante' ( Citrus sinensis L. Osbeck) was the only sweet orange accession with a divergent SNPs genotype, which corroborates the hypothesis of a hybrid origin for this accession. Although the STSs analyzed represent randomly sampled genomic sequences, they provided consistent information about the level of polymorphism and showed the potential of SNPs markers for characterization and phylogenetic studies.

Effect of temperature and leaf wetness duration on infection of sweet oranges by Asiatic citrus canker

Asiatic citrus canker, caused by Xanthomonas smithii ssp. citri, formerly X. axonopodis pv. citri, is one of the most serious phytosanitary problems in Brazilian citrus crops. Experiments were conducted under controlled conditions to assess the influence of temperature and leaf wetness duration on infection and subsequent symptom development of citrus canker in sweet orange cvs Hamlin, Natal, Pera and Valencia. The quantified variables were incubation period, disease incidence, disease severity, mean lesion density and mean lesion size at temperatures of 12, 15, 20, 25, 30, 35, 40 and 42 degrees C, and leaf wetness durations of 0, 4, 8, 12, 16, 20 and 24 h. Symptoms did not develop at 42 degrees C. A generalized beta function showed a good fit to the temperature data, severity being highest in the range 30-35 degrees C. The relationship between citrus canker severity and leaf wetness duration was explained by a monomolecular model, with the greatest severity occurring at 24 h of leaf wetness, with 4 h of wetness being the minimum duration sufficient to cause 100% incidence at optimal temperatures of 25-35 degrees C. Mean lesion density behaved similarly to disease severity in relation to temperature variation and leaf wetness duration. A combined monomolecular-beta generalized model fitted disease severity, mean lesion density or lesion size as a function of both temperature and duration of leaf wetness. The estimated minimum and maximum temperatures for the occurrence of disease were 12 degrees C and 40 degrees C, respectively.

Feeding ecology of a selective folivore, the thin-spined porcupine (Chaetomys subspinosus) in the Atlantic forest

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

Effects of G a3 dosis associated with organosilicone on sweet oranges in Brazil

GA3 was tested in sweet oranges 'Pera' and 'Hamlin' for delay the picking time without loosening of fruit quality for processing. Hamlin is the firths cv processed in Brazil and Pera is a mid season cv and extending their period of processing is important. Two experiments were made at the Citrus Experimental Station during 1996 season. The treatments are 5 ppm of GA 3 + 0,05% Silwett L-77® (organosilicone), 10 ppm of GA 3 + 0,05% of Silwet L-77®, 20 ppm of GA3 + 0,1% Herbitensil® (Noniphenoloxietilate 40%m/v + isopropilic alcohol 15% m/v) and control, repeated 7 times for Hamlin and 8 times for Pera, with one tree each parcel. The treatments were applied in May 1996, at the stage of greenish yellow colour of the fruits. Evaluations were made each 20 days interval till the final picking, It was analysed fruit quality and retention force for picking and puncture resistance. The results showed no differences for fruit quality of Hamlin from July to mid September and for Pera till September. After some differences occurred. The GA3 treatments were effectives in maintain the fruit retention force for both cvs for 120 days after application. In relation to fruit puncture resistance the treatments with GA3 differed of the control for both cvs, accordingly with the doses and mixtures. The colour index was better maintained with 5ppm of GA3 plus 0,05% of Silwet L-77®. The total fruit production did not differ for both cultivars.

Distribution of Xylella fastidiosa in citrus rootstocks and transmission of citrus variegated chlorosis between sweet orange plants through natural root grafts

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Susceptibility of tangerines to citrus variegated chlorosis

Citrus variegated chlorosis (CVC), a citrus disease first discovered in Brazil in 1987, is caused by the bacterium Xylella fastidiosa and transmitted by sharpshooters and budwood. Since the disease affects almost all sweet orange cultivars, it has become one of the most serious problems for Brazilian citriculture. To evaluate their resistance to CVC disease, fifteen tangerines or mandarins (C. reticulata Blanco) and their hybrids were grafted on Rangpur lime (C. limonia Osb.) and inoculated with CVC-contaminated Pera sweet orange (C. sinensis (L.) Osb.) by twig grafting in a greenhouse. Tangerines and their hybrids Wilking, Fortune, Sunki, Ellendale, Orlando tangelo, Nunes clementine, Nova, Sun Shu Sha Kat, Suenkat, and Batangas showed CVC leaf symptoms and gave positive results on enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) (with specific primers for X. fastidiosa), indicating that they are susceptible to CVC. Although X. fastidiosa bacteria were detected by ELISA and PCR in inoculated plants of tangerines Cravo and Oneco, no CVC leaf symptoms were observed on these two cultivars, suggesting that they are tolerant to the disease. CVC leaf symptoms were not observed and X. fastidiosa was not detected in tangerine Dancy and mandarins Okitsu satsuma and Ponkan after inoculation, showing that they are resistant to the disease.

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.

Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings

Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Potential of Brazilian eugenia myrtaceae - As ornamental and as a fruit crop

Brazilian Myrtaceae comprises several genera of trees and shrubs used for ornamental and fruit production. In addition to the well known Guava, Pitanga and Jaboticaba, other species could be used for fruitculture, due the value and quality of their fruits and adaptation to some climate conditions mainly the subtropical one. Nine species of Eugenia were evaluated at Jaboticabal, located at 48° W and 21° S in São Paulo state in a germplasm bank. The average rain by year is 1431 mm and the temperature 22,2° C at an altitude of 575 m. The species are Eugenia klozschiana Berg. (Pero-do-campo), E. stipitata Mc Vaugh (Araça-boi), E. tomentosa Camb. (Cabeludinha), E. dysentherica DC. (Cagaita), E. brasiliensis Berg. (Grumixama), E.pitanga (Pitanga-anã), E. luchsnathiana Berg. (Pitomba), E. uvalha Camb. (Uvaia) and E. involucrata DC. (Cereja-do-rio-grande). The evaluations comprised tree development, fruit quality and leaf and flower morphological studies. The main results are: the trees of Pera-do-campo and Pitanga-anã are small shrubs of 1 to 2 m height, Araça-boi and Cabeludinha are small trees, 3 to 5 m high, and the other species are tall trees, with 5 to 10 m height. The species adapted well to the subtropical conditions, except for Araça-boi, which is native to the Amazonian region and exhibited a severe fungus disease infection. In relation to fruit quality, all the species had edible fruit, some were sweet and juicy, Cabeludinha, Grumixama, Pitomba, Cereja-do-rio grande and Pitanga-anã, while others had high acidity (Araça-boi, Pera-do-campo, Cagaita and Uvaia and were more suitable for processing. Simple, single leaves were characteristic of all species, but with different sizes and shapes., With the addition of color, smell and other characteristics, leaf size and shape were useful for comparative classification. Flower components and structure are described also.

Seletividade de alguns agrotóxicos em uso na citricultura ao ácaro predador Neoseiulus californicus (McGregor) (Acari: Phytoseiidae)

This work was carried out to determine the residual toxicity of the main pesticides used in citrus, on Neoseiulus californicus (McGregor) under laboratory conditions. For this, it was used the residual contact bioassay. Citrus leaves of the variety Pêra were sprayed in a Potter tower. The residual action was evaluated at 2 hours and 1; 3; 5; 7; 10; 14 e 21 days after treatment. Ten adult females of N. californicus were transferred to each ring, together with an enough amount of Tetranychus urticae nymphs and adults to feed the predator. Mortality evaluations were performed at 72 hours after treatment. Bioassays indicate that the pesticides: acrinathrin, deltamethrin, dinocap, sulphur, fenpropathrin, fenbutatin oxide and propargite were innocuous to N. californicus. On the other hand, abamectin, azocyclotin e cyhexatin caused mortalities of 29.8; 32.0 e 34.1%, respectively, two hours after transference. Dicofol, pyridaben and chlorfenapyr were extremely toxic to the predator mite, causing 100% of mortality two hours after the application. This way, to this population of N. californicus, the releases can be performed safely 3 days after the application, except for cyhexatin (5 days), dicofol and pyridaben (14 days) and chlorfenapyr (21 days), without risk of significant adult mortality rates due to the application of pesticides.

Aspectos técnicos e econômicos da poda e do controle químico de Brevipalpus phoenicis no manejo da leprose dos citros

The objective of this study was to evaluate different citrus leprosis management tactics during seven seasons, based in pruning and acaricide applications, considering technical and economic aspects of each tactic. The trial was conducted from October 2003 to August 2010 in an orange plantation of Pera cv. located in the municipality of Reginópolis-SP, Brazil. The plants of citrus used were12 years old and grafted onto Cleopatra tangerine. The experimental design used was randomized blocks, in a factorial scheme, made up of factors type of pruning (A), with six levels: (1) drastic pruning, (2) intermediate pruning without leprosis lesions, (3) intermediate pruning with leprosis lesions (4) light pruning, (5) without pruning and (6) replant; acaricide applications factor (B), with three levels: (1) without acaricides applications, (2) with lime sulfur applications and (3) spirodiclofen or cyhexatin applied in rotation; pruning factor to remove leprosis symptomatic branches (C), with two levels: (1) with pruning for removal, (2) without removal pruning. The combination of factors, with respective levels (6 x 3 x 2), resulted in 36 treatments that were repeated four times, with each parcel being made up of three plants in a row. After seven years, it was observed that the types of the pruning and remove of leprosis symptomatic branches used as single management tactic, is not sufficient to leprosis control. Therefore, the results demonstrated that for leprosis management, it is essential the association between tactics, especially the control mite vector. For ensuring the citrus production economically, the use of acaricides highly efficient is essential in B. phoenicis control. The recommendation of the type of pruning should be in function of the leprosis incidence and severity in the orchard. In orchards with low leprosis incidence and severity the light pruning is more appropriate, because it is efficient and ensures greater financial balance. However, in orchards with high leprosis incidence and severity it is indicated the severe pruning for reducing or eliminating inoculum source. Between the severe prunings, the intermediate pruning with leprosis lesions is the most relevant, because the financial return will be faster. Replant is indicated only for young orchards, because does not imply in changes onto cultural practices.

Physico-chemical characterization of seed oils extracted from oranges (Citrus sinensis)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)