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.

Saprophytic colonization of citrus twigs by Diaporthe citri and factors affecting pycnidial production and conidial survival

Melanose, caused by Diaporthe citri, produces reddish brown lesions on the fruit, leaves, and twigs of citrus trees, and greatly reduces the marketability of fresh fruit. Most of the inoculum is produced in pycnidia on dead twigs in the tree canopy, which exude large numbers of conidia in slimy masses. In this study, detached twigs inoculated with conidia were readily colonized and produced large numbers of pycnidia within 30 to 40 days when they were soaked 3 to 4 h on alternate days. Conidial production was measured by wetting twigs in a rain tower periodically and collecting the conidia in the runoff water. Production began after 80 days and continued for nearly 300 days. In other experiments, production of mature pycnidia on detached twigs was greatest at 94 to 100% relative humidity (RH) and at 28 degrees C. Low RH and temperature, however, favored survival of conidia in exuded masses on twigs. In the field, colonization of detached twigs by D. citri was high in rainy season, moderate in spring and early fall, and minimal in late fall and winter. Twig colonization was positively related to the number of rain days and average temperature, but not to total rainfall. In another experiment, inoculated twigs placed in the tree canopy developed pycnidia and then produced conidial masses for about 200 days. D. citri is a serious pathogen, but a weak parasite, that survives primarily by colonization and reproduction on dead twigs.

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.

Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type

The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5' regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 3'-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

Pectin lyase from Aspergillus giganteus: Comparative study of productivity of submerged fermentation on citrus pectin and orange waste

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

Nutrient digestibility of degermed, dehulled corn, citrus pulp, and soy protein concentrate by barrows

This study was carried out to determine apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of DM, CP, GE, and their respective digestible content of degermed dehulled corn (Zea mays), citrus pulp, and soy (Glycine max) protein concentrate by pigs using the difference method. Thirty-two barrows (28.1 +/- 1.6 kg of BW) were fed a corn-soybean meal basal diet or 1 of 3 diets formulated by replacing 30% of the basal diet with 30% of 1 of the test feedstuffs for 11 d. Chromic oxide (0.3%) was included in the diets. Feces were collected from days 7 to 11 by grab sampling and ileal digesta were collected after pigs were slaughtered on day 12. The AID of DM and AID and ATTD of GE of degermed corn (77.4, 88.7, and 77.7%) were greater (P < 0.05) than those observed in citrus pulp (50.3, 86.5, and 55.8%) and in soy protein concentrate (63.5, 85.1, and 59.4%), which did not differ (P > 0.05). The ATTD of CP, total digestible CP, and total DE of soy protein concentrate (87.5%, 500 g/kg, and 3739 kcal/kg) were higher (P < 0.05) than the values in degermed corn (81.7%, 57.5 g/kg, and 3330 kcal/kg), which were greater (P < 0.05) than those in citrus pulp (60.5%, 39.5 g/kg, and 3223 kcal/kg). Total and ileal digestible DM, AID of CP, and ileal DE of degermed corn (782 g/kg, 673 g/kg, 70.7%, and 2913 kcal/kg) and soy protein concentrate (778 g/kg, 570 g/kg, 78.7%, and 2878 kcal/kg) were similar (P > 0.05) and greater (P < 0.05) than those in citrus pulp (737 g/kg, 436 g/kg, 50.6%, and 2081 kcal/kg). Ileal digestible CP of degermed corn (49.8 g/kg) and citrus pulp (33.0 g/kg) did not differ (P > 0.05) but were smaller (P < 0.05) than the value found in soy protein concentrate (434 g/kg). The DM and energy from degermed corn are more efficiently digested by the pig than those from soy protein concentrate and citrus pulp. Soy protein concentrate was the best protein source evaluated in this study.

Fate of Some New Fungicides (Cyprodinil, Fludioxonil, Pyrimethanil, and Tebuconazole) from Vine to Wine

The fate of four new fungicides (cyprodinil, fludioxonil, pyrimethanil, and tebuconazole) from the treatment on vine to the production of wine was studied. The influence of clarifying agents (bentonite, charcoal, potassium caseinate, gelatin, and polyvinylpolypyrrolidone) on residue concentrations in wine was also studied. The fungicide residues on grapes showed different decay rates after treatment, with first-order kinetics and half-lives ranging from 8 to 57 days. Grape processing into wine caused considerable residue reduction with cyprodinil (ca. 80%), fludioxonil (ca. 70%), and tebuconazole (ca. 50%) and no reduction with pyrimethanil. The two wine-making techniques employed (with and without maceration) had the same influence on the residue concentrations in wine, except for fludioxonil which showed maximum residue reduction with vinification with maceration. Among the clarifying agents tested, only charcoal showed effective action on the elimination of residue content in wine, proving complete elimination, or almost, of fungicide residues.

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.

Aspects of IFP for citrus production in Brazil

Brazilian citriculture represents about 25% of the total world citrus production with an area of 851,518 ha and a total production of more than 17 million tons in 1996. Besides its importance to the brazilian economy, represented by more than 1 billion US $ by year from FCOJ exportation, the citriculture has problems related to low productivity, due to several cultural practices and management. The productivity would be improved by an IFP system. The main problems are related to soil, as poor conservation, use of poor drained soils and bad preparation for planting; diseases-canker, CVC, leprosis, Phytophtora gummosis and other fungus diseases; pests - mites, scales, nematodes and others; the use of disease free and improved scion and rootstocks propagative material is usual; cultural practices - as nutrition, irrigation, wind breaks, weed control, pruning, replant and others, as density planting could be improved. Some possible solutions will be discussed for improving the brazilian citrus productivity and quality by IFP, based on research made.

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.

Adubação verde em Citrus: Influência na porosidade e retenção de água de um latossolo vermelho-escuro do noroeste do estado de São Paulo

The objective of this research was to study the porosity, bulk density and retention of water of an Oxisol, located in the Northwestern region of Sn̄o Paulo state, Brazil. The soil was cultivated with Citrus sp., to which green manure was applied between rows for three years. Each of six species of green manure crops (Crotalaria juncea L., Mucuna deeringiana Steph. & Bart., Canavalia ensiformis L. DC., Cajanus cajan L., Lablab purpureum L. and Ricinus communis L.) were seeded for three years (1995, 1996 and 1997) between Citrus rows, plus a treatment with a mix of all six species and a control (natural regrowth af vegetation). The experimental design was a randomized complete block design, with four replications for each of the eight treatments. Water retention, microporosity, macroporosity, total porosity and bulk density were analyzed in the beginning (1995) and end (1997) of the experiment, at three depth ranges (0-0.10; 0.10-0.20 and 0.20-0.40m). We concluded that there were statistically significant differences for bulk density, macroporosity, total porosity and retention of water among the different soil depth ranges; there were no significant differences among treatments though.

Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus

Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum, produces orange-brown lesions on petals and results in premature fruit drop and the retention of calyces. C. gloeosporioides is common in groves and causes postharvest anthracnose on fruit. Both diseases are controlled effectively by the fungicide benomyl in research fields and commercial orchards. Highly sensitive and resistant isolates of C. gloeosporioides were found, whereas all isolates of C. acutatum tested were moderately resistant. In preliminary studies conducted in vitro with three isolates of each, mycelial growth of sensitive isolates of C. gloeosporioides was inhibited completely by benomyl (Benlate 50 WP) at 1.0 μg/ml, whereas resistant isolates grew well at 10 μg/ml. Growth of all isolates of C. acutatum was inhibited by about 55% at 0.1 μg/ml and by 80% at 1.0 μg/ml. Spore germination of C. acutatum was inhibited more at 0.1 μg/ml than at 1.0 μg/ml or higher concentrations. In all, 20 isolates of C. acutatum from 17 groves and 20 isolates of C. gloeosporioides from 7 groves were collected from locations with different histories of benomyl usage in São Paulo, Brazil, and Florida, United States. Benomyl at 1.0 μ.g/ml completely inhibited growth of 133 isolates of C. gloeosporioides, with the exception of 7 isolates that were highly resistant to the fungicide, whereas all isolates of C. acutatum were only partially inhibited at 0.1 and 1.0 μg/ml. Analysis of variance indicated that the sensitivity of the isolates of C. acutatum was not affected by benomyl usage or grove of origin, and country of origin had only minor effects. No highly resistant or sensitive isolate of C. acutatum was recovered. Partial sequencing of the β-tubulin gene did not reveal nucleotide substitutions in codons 198 or 200 in C. acutatum that usually are associated with benomyl resistance in other fungi.

Attractiveness of citrus pulp and orange albedo extracts to Atta sexdens rubropilosa (Hymenoptera: Formicidae)

The present study aimed to compare the attractiveness of industrial citrus pulp with the handmade orange albedo to the workers of Atta sexdens rubropilosa. For this, filter paper fragments were impregnated with organic extracts obtained through chemical extraction and sequential fractioning with hexane and dichloromethane and offered to different field nests. It was verified that the industrial citrus pulp extract is as good as the handmade orange albedo extract. This preference is discussed keeping in mind the chemical, behavioral and nutritional factors.