Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Pratylenchus jaehni sp n. from citrus in Brazil and its relationship with P-coffeae and P-loosi (Nematoda : Pratylenchidae)

A lesion nematode population infecting citrus in the state of São Paulo, Brazil is described and named Pratylenchus jaehni sp. n. Biological, molecular and morphological characteristics of this new species are compared with those of the morphologically similar P. coffeae and P. loosi. Results of mating experiments showed that R jaehni is reproductively isolated from P coffeae. Molecular (D2/D3 DNA sequences) dissimilarities among P. jaehni sp, n., P. coffeae and P. loosi were documented in a previous study. The morphology of seven R coffeae populations from tropical America and eastern Java and a P loosi population from Sri Lanka is used for comparison with the morphology of P. jaehni sp. n. Pratylenchus jaehni differs from R coffeae and P. loosi by only a few morphological ;characters of the females. The mean values of stylet length, stylet knob height, and vulva position are smaller (less than or equal to15 vs greater than or equal to15 mum, less than or equal to2.7 vs greater than or equal to2.7 mum, less than or equal to79 vs greater than or equal to79%) than those in P coffeae and P loosi. The tail terminus is usually subhemispherical and smooth in P jaehni sp. n.. whereas it is commonly truncate and indented in most P. coffeae populations and bluntly or finely pointed in P. loosi. Because of the morphological similarities among P. jaehni sp. n., P. coffeae and P. loosi, examination of at least ten specimens is required to obtain a reliable diagnosis based on morphology. Nineteen morphometric parameters for P. jaehni sp. n. and P. coffeae ranged from 0-13% smaller in fixed than in live specimens.

Partial purification and characterization of pectin methylesterase from orange (Citrus sinensis) cv. Pera-rio

The enzyme pectin methylesterase (PME) from orange was extracted and partially purified by filtration on Sephadex G-100. The extraction buffer for orange PME was borate-acetate containing 0.4 M NaCl. Orange PME showed optimum pH at 8.0 and optimum temperature at 50C. The PME enzyme was completely inactivated after 1 min of incubation at 90C. The specific activity increased in the presence of 0.15 M NaCl or 0.025 M Na2SO4, 0.10 M KCl, 0.025 M K2SO4, 0.05 and 0.1 M NH4Cl. Lithium chloride and Li(2)SO(4)inhibited the enzymatic activity at all concentrations studied. The K-m and V(max)value of PME were 0.36 mg/mL and 5.26 mu mol/mL-mg protein, respectively.

Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain

Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.

Determination of buprofezin, pyridaben, and tebufenpyrad residues by gas chromatography mass-selective detection in clementine citrus

A gas chromatography-mass-selective (GC-MS) detection method to determine buprofezin, pyridaben, and tebufenpyrad on the pulp, peel, and whole fruit of clementines is described. The extraction/partition procedure was performed in one step and no cleanup was necessary with the GC-MS in the SIM-mode pesticide determination. Recovery ranged from 75 to 124% with coefficients of variance ranging between 1 and 13%. The limit of determination was 0.01 mg/kg for all pesticides. The field trials showed a similar degradative behavior for all active ingredients (AI), with a great residue decrease during the first week and stability in the second. Just after treatment buprofezin and tebufenpyrad showed lower residues than the maximum residue limit (MRL) fixed in Italy, while pyridaben was below the MRL after a week.

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.

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.