Breeding, genetic and genomic of citrus for disease resistance

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.

The effects of inter-crop cultivation Between rows of citrus crop on spreading of Guignardia citricarpa Ascospores and in the citrus black spot occurrence

This study highlighted the effect of planting coast-cross grass and forage peanut cv. Amarilis between rows of Natal oranges on spreading of Guignardia citricarpa ascospores and consequent citrus black spot control. Treatments evaluated were: 1- conventional cultivation, free of fungicides; 2- conventional cultivation, using protective fungicides; 3- inter-crop cultivation of coast-cross grass between rows of citrus crops and; 4- inter-cropping cultivation of forage peanut between the rows of citrus crops. Quest Volumetric Spore SystemTM traps were set in order to determine the number of ascospores released. A total of 33 inspections were conducted weekly, from the end of August until early September the following year. A diagrammatic scale was used to determine the severity of the disease as well as the percentage of fruits having a commercial standard. The coast-cross grass was more effective in reducing the number of ascospores produced, whose average statistics were lower than in the conventional treatments, free-fungicides. The inter-crop and conventional cultivation method coupled with fungicide treatment was more effective in reducing the severity of citrus black spot symptoms, and differs statistically from the fungicide-free control method. These methods also resulted in a higher percentage of fruits of a commercial standard, ranging from the 89% through the 91% percentile, and the cultivation, free of fungicides, fell within the 73%.

Monitoring the viability of citrus rootstocks seeds stored under refrigeration

The citrus nursery tree is produced through the bud grafting process, in which rootstock is usually grown from seed germination. The objective of this research was to evaluate, in two dissimilar environmental conditions, the viability and polyembryony expression of five citrus rootstocks seeds stored in different periods under refrigeration. The rootstock varieties evaluated were: Rangpur lime (Citrus limonia Osb. cv. Limeira), Trifoliate orange (Poncirus trifoliata Raf. cv. Limeira), Citrumelo (P. trifoliata x C. paradisi Macf. cv. Swingle), Sunki mandarin (C. sunki Hort. ex Tanaka) and Volkamer lemon (C. volkameriana Ten. & Pasq. cv. Catania 2). The experimental design was the randomized blocks in a 11 x 5 x 2 factorial scheme, evaluating from time zero to the tenth month of storage, the five varieties of rootstock in two environments: germination and growth B.O.D type chamber (Biological Oxygen Demand - Eletrolab Brand Model FC 122) at 25 °C; and greenhouse seedbed with partial temperature control (22 °C to 36 °C) and humidity control (75-85%). The plot had 24 seeds in four replicates, using trays with substrate in greenhouse and Petri dishes with filter paper in B.O.D. chamber. The seed germination rate and polyembryony expression were evaluated monthly. It was concluded that Trifoliate and Citrumelo Swingle seeds can be stored for up to seven months, while Volkamer lemon, Rangpur lime and Sunki seeds can be stored for up to ten months. The polyembryony expression rate was slightly higher when measured in greenhouse than in B.O.D. chamber and remained stable in both environments until the seventh month, from which dropped sharply. Citrumelo Swingle seeds expressed the highest polyembryony rate (18.8%), followed by Rangpur lime and Volkamer lemon (average value of 13.7%), Sunki (9.4%) and Trifoliate (3.2%). Despite some differences among varieties, the viability of rootstock stored seeds can be monitored either in the greenhouse or in B.O.D. germination chamber, the latter being the faster and more suitable method.

Occurrence of Helicoverpa armigera (Hübner, 1808) on citrus in the state of Sao Paulo, Brazil

The occurrence of Helicoverpa armigera (Hübner, 1808) was first reported in citrus orchard in the state of São Paulo (SP). High infestation levels of H. armigera were observed in October 2012, in the city of Botucatu, SP. The larvae was fed of all parts of the plants. The injuries on the leaves caused drastic reduction in the leaf area and the fruits attack occurred from an early stage of development to the ripe fruit. Thus, the first occurrence of H. armigera in this citrus culture adds to the list of hosts of this pest, and is of great importance, because it confirms H. armigera potential dispersion and polyphagia.

Comportamento de híbridos de citros em relação à infecção natural pelo Citrus tristeza virus e à presença de sintomas de descamamento eruptivo

O Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura vem gerando híbridos para utilização como porta-enxertos, que necessitam ser avaliados em relação ao comportamento frente à infecção natural por isolados locais de Citrus tristeza virus (CTV) e à presença de sintomas de descamamento eruptivo (BahiaBarkScaling disease - BBS). Este trabalho apresenta resultados da avaliação do comportamento de 141 híbridos (sob a forma de pés-francos ou enxertados) estabelecidos na área experimental da Embrapa Mandioca e Fruticultura, no Recôncavo Sul da Bahia. Foram avaliadas a presença e a severidade de sintomas de caneluras e descamamento por meio de escala de notas. Para detectar a presença do CTV, foi utilizado o método sorológico de ELISA indireto e RT-PCR. Os híbridos avaliados foram classificados como imunes, tolerantes e intolerantes ao CTV. A maioria dos híbridos que apresentaram sintomas de BBS tem uma tangerineira como parental.

Flutuação populacional e dependência espacial de Aleurocanthus woglumi Ashby, 1915 (Hemiptera: Aleyrodidae) em Citrus latifolia

A mosca-negra-dos-citros, Aleurocanthus woglumi, apresenta elevada capacidade de dispersão e adaptação a diversas condições climáticas, além de grande potencial reprodutivo. O presente trabalho teve como objetivo avaliar a flutuação populacional e caracterizar o padrão de dependência espacial de A. woglumi em um pomar de lima- ácida Tahiti (Citrus latifolia, Tanaka), no município de São José de Ribamar, Estado do Maranhão. Um sistema de posicionamento global (GPS) e um sistema de informação geográfica (SIG) foram usados para localizar os pontos das amostras, caracterizar o padrão de distribuição, calcular a área de agregação e elaborar mapas de distribuição dos adultos de mosca-negra-dos-citros dentro do pomar. O número médio total de moscas negras foi maior durante a estação chuvosa e observaram-se correlações significativas entre as variáveis precipitação pluvial, umidade relativa do ar e número total de adultos. A distribuição espacial A. woglumi no pomar é agregada, ajustando-se os variogramas calculados ao modelo esférico nas estações seca e chuvosa. Os insetos mostraram uma área de agregação média de 162.092 m² na estação chuvosa e de 9.615 m² na estação seca. Para se obter uma estimativa confiável de populações de mosca-negra-dos-citros, pelo menos uma armadilha deve ser usada a cada 17 hectares na estação chuvosa e uma armadilha por hectare na estação seca.

Atividade antioxidante do extrato de sementes de limão (Citrus limon) adicionado ao óleo de soja em teste de estocagem acelerada

The aim of this work was to evaluate antioxidant activity of lemon seeds added to soybean oil, submitted to accelerated incubator-storage test and to determine its synergistic effect with the synthetic antioxidant TBHQ. The treatments Control, TBHQ (50 mg/kg), LSE (2,400 mg/kg Lemon Seed Extract), Mixture 1 (LSE + 50 mg/kg TBHQ) and Mixture 2 (LSE + 25 mg/kg TBHQ) were prepared and subjected to the accelerated incubator-storage test at 60 ºC for 12 days; samples were taken every 3 days and analyzed regarding peroxide value and conjugated dienes. The results showed that antioxidant activity of the tested treatments were: TBHQ = Mixture 1 = Mixture 2 > LSE > Control.

Composição química e atividade anticolinesterásica de uma fração ativa do extrato de folhas de Citrus limon (L.) Burm

This paper describes the chemical composition and acetylcholinesterase inhibition of an active chromatographic fraction of the EtOAc extract of Citrus limon leaves. The composition of the active fraction presented a mixture of two coumarins, 5,8-dimethoxy-psoralen and 5,7- dimethoxycoumarin, identified by ¹H and ¹³C NMR data analysis, including DEPT, COSY, HMBC and HSQC experiments. It was also demonstrated that this mixture presents qualitative and quantitative AChE inhibition. In vitro studies indicated a CE50 value of 340 µg/mL with 95% of confidence. In vivo studies (10 and 25 mg/kg) revealed inhibition of 30.09 and 30.06% of AChE activity in relation to neostigmine, respectively.

TRIAGEM METABÓLICA POR PKS E NRPS EM ACTINOBACTÉRIAS ENDOFÍTICAS DE Citrus reticulata

Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains.

Woody Gall symptoms in different citrus species and varieties artificially colonized by the citrus brown aphid Toxoptera citricidus

Seedlings of 41 different citrus species and varieties were massively colonized with the citrus brown aphid Toxoptera citricidus, obtained from Pêra sweet orange (Citrus sinensis) trees, presenting symptoms of the "Capão Bonito" complex of the Citrus tristeza virus (CTV). The objective was to evaluate resistance or tolerance of the varieties to that virus complex, but even after eight months of inoculation no stem pitting was observed in the plants. Otherwise, the presence of galls similar to those induced by the vein enation-woody gall disease was observed in 73% of the plants of Volkamer Palermo (Citrus volkameriana), 60% of the Volkamer Catania 2, 2% of the Rangpur Lime D.22.30 (Citrus limonia), 13% of the Volkamer Australian Red, the Rangpur Lime hybrid, the Orlando tangelo (Citrus reticulata x Citrus paradisi) and the Florida Rough lemon (C. jambhiri), and 7% of the Carrizo citrange (Poncirus trifoliata x Citrus sinensis). The highest incidence and the largest gall size were observed in the Palermo Volkamer showing that this clone would be the most suitable to be used as an indicator plant in biological indexing tests for the disease. There are no previous reports in the literature about the occurrence of woody galls in Orlando tangelo and Carrizo citrange.

Reação de porta-enxertos híbridos ao Citrus tristeza virus

A tristeza causada pelo vírus da tristeza dos citros (Citrus tristeza virus, CTV) é uma das principais viroses dos citros (Citrus spp.) no Brasil. Alguns autores têm utilizado a intensidade de caneluras produzidas nos ramos para selecionar plantas com resistência ao vírus. Neste trabalho foi avaliada a reação de porta-enxertos híbridos, provenientes do programa de melhoramento genético de citros da Embrapa Mandioca e Fruticultura ao CTV e elaboradas duas escalas, uma fotográfica e outra diagramática, para quantificação de resistência ao CTV. Entre os porta-enxertos avaliados, a maioria apresentou poucas caneluras, sendo portanto considerados resistentes à tristeza. Verificou-se a manutenção da resistência ao vírus nos híbridos produzidos a partir de progenitores que possuíam algum nível de resistência.

Geostical analysis of spatial dynamics of Citrus Variegated Chlorossis

The spatial dynamics of Citrus Variegated Chlorosis (CVC) was studied in a five-year old commercial orchard of 'Valencia' sweet orange (Citrus sp.) trees, located in the northern region of the state of São Paulo, Brazil. One thousand trees were assessed in 25 rows of 40 trees, planted at 8 x 5 m spacing. Disease incidence data were taken beginning in March 1994 and ending in January 1996, at intervals of four to five months. Disease aggregation was observed through the dispersion index analysis (Ib), which was calculated by dividing the area into quadrants. CVC spatial dynamics was examined using semivariogram analysis, which revealed that the disease was aggregated in the field forming foci of 10 to 14 m. For each well-fitted model, a kriging map was created to better visualize the distribution of the disease. The spherical model was the best fit for the data in this study. Kriging maps also revealed that the incidence of CVC increased in periods during which the trees underwent vegetative growth, coinciding with greater expected occurrence of insect vectors of the bacterium in the field.

Nematode community, trophic structure and population fluctuation in soybean fields

Temporal (monthly in three fields for 12 months) and spatial (once in 23 fields during March-April) samplings were conducted in the major soybean (Glycine max)-growing region of the Brazilian Federal District. Fifty-three nematode genera were found in both samplings, but 13 were detected only by the temporal sampling, and one only by the spatial sampling. Fifty-three percent were plant-parasites, 35% were bacterivores, and about 12% were fungivores, predators and omnivores constituted the community that was dominated by the genera Helicotylenchus (40% of total abundance), Acrobeles (15%), Cephalobus (7.6%), Meloidogyne(5.6%) and Pratylenchus (4.9%). Heterodera glycines was not found in this study. There were no differences in ten ecological measurements [Ds, H', Es, T, FF/BF, (FF+BF)/PP, MI, PPI, mMI, and Dorylaimida (%)] between the two sampling types, but differences in indexes d and J'. Plant parasite populations dropped at the end of the crop cycle, remained at low levels during the dry season and the seedling period, then increased again in the crop-growing season. Fungivores maintained their low populations throughout the year, increasing only in June and July, the post-harvest period, when soil fungi decomposed root tissue. The population of bacterivores slightly declined during the dry season and the initial rainy season, but peaked in the middle of the rainy season, apparently associated with soil humidity. In the five most abundant nematodes, those of Acrobeles and Pratylenchus were more populous in wet soils, Cephalobus and Meloidogyne adapted well in dry soils, but Helicotylenchus survived abundantly in a wide range of soil moisture.

Caracterização de isolados de Phytophthora patogênicos a Citrus sinensis no Estado de Alagoas

Nove isolados de Phytophthora obtidos da rizosfera de laranjeiras (Citrus sinensis) infetadas, em Alagoas, foram caracterizados com base na morfologia da colônia, morfologia e morfometria das estruturas reprodutivas, e no crescimento em diferentes temperaturas. Todas as culturas apresentaram-se heterotálicas; esporângios papilados, ovóides ou subesféricos, medindo 24,6 - 78,7 µm de comprimento (média=49,1µm) x 16,4 - 49,2 µm de largura (média=33,3 µm), com uma relação comprimento/largura de 1,1 - 2,3 (média=1,5); clamidósporos principalmente terminais, apresentando 13,1 - 45,9 µm de diâmetro (média=27,3 µm). Oogônios globosos, com 14,8 - 34,4 µm de diâmetro (média=26,4 µm) contendo oósporos apleuróticos, medindo 11,5 - 29,5 µm de diâmetro (média=22,7 µm). Anterídios em posição anfígena, medindo 6,6 - 16,7 µm de comprimento (média= 10,6 µm) e 8,2 - 16,7 µm de largura (média=12,0 µm). O maior crescimento micelial ocorreu entre 25 e 30 ºC em meio de cenoura-ágar modificado. Todos os isolados cresceram a 35 ºC e foram patogênicos às mudas de limão (Citrus limonia) 'Cravo' e aos frutos de laranja 'Pêra'. Todos os isolados foram identificados como P. nicotianae (= P. parasitica), pertencentes ao tipo compatível A1.