Antioxidant and antifungal activities of Camellia sinensis (L) Kuntze leaves obtained by different forms of production / Atividade antioxidante e antifúngica das folhas de Camellia sinensis (L) Kuntze, obtidas por diferentes formas de produção

Abstract The antioxidant and anticandidal activities of leaves obtained from Camellia sinensis by non-fermentation (green and white teas), semi-fermentation (red tea) and fermentation method (black tea) were investigated. It was evaluated the total phenolic content by Folin-Ciocalteau assay; antioxidant capacities were evaluated in vitro using DPPH and ABTS radicals, hypochlorous acid and superoxide anion scavenger assays, induced hemolysis, lipid peroxidation by conjugated diene formation and myeloperoxidase activity. Anticandidal activity was performed on three strains of Candida spp. The results showed that non-fermented teas have a higher concentration of phenolic compounds, and then presented the best inhibitory activity of AAPH-induced hemolysis, the best inhibition of conjugated diene formation and more pronounced antioxidant activity in all tests. The highest anticandidal activity was obtained from fermented tea, followed by non-fermented tea. These results indicate that the antioxidant activity demonstrated has no direct relation with the anticandidal activity.

Resumo A atividade antioxidante e antifúngica das folhas obtidas da Camellia sinensis pelos métodos de não-fermentação (chás verde e branco), semi-fermentação (chá vermelho) e fermentação (chá preto) foram investigadas. Foi avaliado o conteúdo total de compostos fenólicos pelo método de Folin-Ciocalteau; a capacidade antioxidante foi avaliada in vitro usando os radicais artificiais DPPH e ABTS, o ácido hipocloroso, ensaios do ânion superóxido, hemólise induzida, peroxidação lipídica por formação de dienos conjugados e atividade da Mieloperoxidase. A atividade antifúngica foi obtida sobre três cepas de Candida spp. Os resultados obtidos mostram que os chás não fermentados apresentam a maior concentração de compostos fenólicos e também, apresentam a melhor atividade inibitória, sobre hemólise induzida por APPH, sobre a formação de dienos conjugados e a mais pronunciada atividade antioxidante sobre todos os testes. A maior atividade antifúngica foi obtida pelo chá fermentado, seguido pelo semi-fermentado e não-fermentados. Os resultados obtidos demonstram que a atividade antioxidante observada não apresenta relação com a atividade antifúngica.

Antioxidant and antifungal activities of Camellia sinensis (L.) Kuntze leaves obtained by different forms of production.

The antioxidant and anticandidal activities of leaves obtained from Camellia sinensis by non-fermentation (green and white teas), semi-fermentation (red tea) and fermentation method (black tea) were investigated. It was evaluated the total phenolic content by Folin-Ciocalteau assay; antioxidant capacities were evaluated in vitro using DPPH and ABTS radicals, hypochlorous acid and superoxide anion scavenger assays, induced hemolysis, lipid peroxidation by conjugated diene formation and myeloperoxidase activity. Anticandidal activity was performed on three strains of Candida spp. The results showed that non-fermented teas have a higher concentration of phenolic compounds, and then presented the best inhibitory activity of AAPH-induced hemolysis, the best inhibition of conjugated diene formation and more pronounced antioxidant activity in all tests. The highest anticandidal activity was obtained from fermented tea, followed by non-fermented tea. These results indicate that the antioxidant activity demonstrated has no direct relation with the anticandidal activity.

First Report of Powdery Mildew Caused by Erysiphe platani on Platanus × acerifolia in Rio Grande do Sul, Brazil.

Platanus × acerifolia (Aiton) Willd. (London planetree) is a tree commonly used as an ornamental and in the furniture industry. In the summer of 2013, powdery mildew was observed on shoots of P. × acerifolia plants in the cities of Pelotas and Canela (State of Rio Grande do Sul, Brazil). Voucher specimens (n = 2) were deposited in the Phytopathological Museum Manoel Alves Oliveira at Federal University of Pelotas. Dense white powdery masses of conidia and mycelium were observed on leaves (abaxial and adaxial surfaces), petioles, and young stems. Leaves with high disease severities (≥70%) were deformed with curved edges to the adaxial side, and they often died. Mycelia were superficial with lobed appressoria. Conidiophores were straight, sometimes curved at the base, unbranched, cylindrical, 98 to 236 µm long (137.3 ± 41.2 µm) and composed of a cylindrical foot cell 49 to 102 µm long (66.9 ± 19.5 µm) and 4.4 to 6.4 µm wide (5.3 ± 0.8 µm) followed by two to four cells. Conidia were produced singly or in short chains (two to three), without distinct fibrosin bodies, ellipsoid to ovoid and measuring 24 to 37 µm long (29.5 ± 3.2 µm) and 12 to 19 µm wide (15.2 ± 1.4 µm), often with a wrinkled appearance. Primary conidia had truncate bases and rounded apex while both base and apex were truncated in secondary conidia. Germ tubes were produced apically (pseudoidium type). Chasmothecia were not observed. Genomic DNA was used to amplify the internal transcribed spacer (ITS) region using the ITS1 and ITS4 primers. The resulting sequence (602 bp) was deposited (Accession No. KF499270) in GenBank. BLASTn searches revealed similarity of 100 and 99% with Erysiphe platani from P. orientalis L. (Accession No. JQ365943.1) and P. occidentalis L. (Accession No. JX997805.1), respectively. Phylogenetic analysis placed our sequence in a clade (99% bootstrap support) which included only other E. plantani sequences. In short, morphological and molecular approaches allowed us to identify the infecting fungus as E. platani. For Koch's postulates, 10 detached leaves were inoculated (10 to 15 conidia cm2) on their adaxial surface using an eyelash brush. Non-inoculated leaves served as control. All leaves were kept inside trays with petiole immersed in humidified cotton and maintained at 25 ± 1°C. Symptoms identical to those of the original leaves were observed 6 to 8 days after inoculation, whereas the control leaves remained symptomless. Although E. platani has been previously reported on P. × acerifolia in the city of Poços de Calda, state of Minas Gerais, Brazil (1) and on P. occidentalis in Korea (2), to our knowledge, this is the first record of E. platani on P. × acerifolia in Rio Grande do Sul, Brazil. References: (1) E. M. Inokuti et al. New Dis. Rep. 15:38, 2007. (2) Y. J. La and H. D. Shin. Plant Dis. 97:843, 2013.

Gray Mold Caused by Botryotinia fuckeliana on Edible Pods of Pea in Brazil.

Gray mold on edible pods of snow pea (Pisum sativum Lam. [Fabaceae]) was observed in greenhouse-cultivated pea (cvs. Luana Gigante and Gigante Flor Roxa) in the city of Pelotas (Rio Grande do Sul, Brazil) in September and October 2012. The incidence of diseased pods was high (∼25% of immature pods) after up to 3 cloudy and rainy days that hindered the ventilation inside the greenhouse resulting in high relative humidity. Infection occurred first on senescing petals adhered to the forming pods, leading to pod abortion or rotting that began at the contact site with the infected petal. The first symptoms on pods included water soaked tissue that quickly turned light brown and progressed to necrosis. Conidia and conidiophores produced on profuse gray mycelium could be easily seen on infected tissue 2 to 3 days after the appearance of symptoms. Conidiophores were smooth-walled, 400 µm to over 1.5 mm long, hyaline to pale brown, and branched in their upper part; each branch ended with a hemispherical or spherical swelling, 5 to 9 µm in diameter with minute sterigmata. Macroconidia were globose, ellipsoidal, smooth, hyaline to pale brown, usually with protuberant hila, 7 to 15 × 5 to 9 µm. Microconidia were not observed. On potato dextrose agar (PDA), colonies were fast-growing, white, low, covering entire 10 cm petri plates in 4 to 5 days when they turned gray to brownish-gray. Conidiophores and conidia were often formed in sectors. Shield-like, elliptical, lenticular to irregular, black, 1.5 to 6.0 × 1.0 to 4.0 mm sclerotia developed in 10-day-old colonies incubated at room temperature. Genomic DNA was extracted from conidia, conidiophores, and mycelium and used to amplify both the internal transcribed spacer (ITS) (ITS1-5.8s-ITS2) region and the ß-tubulin gene using the ITS1/4 and Bt2a/b primers, respectively (1,4). The ITS (541 bp) and ß-tubulin (467 bp) sequences were deposited in GenBank under accessions KC683713 and KC683712, respectively. BLASTn searches revealed similarity of 100% (EF207415) and 99% (FQ790278) with Botryotinia fuckeliana (De Bary) Whetzel for the ITS and ß- tubulin sequences, respectively. Based on morphological characteristics and sequence analysis, the pathogen causing pod rot of peas was identified as B. fuckeliana. To fulfill Koch's postulates, 10 unwounded pods of P. sativum 'Luana Gigante' were inoculated by depositing PDA plugs (5 mm) colonized with fungal mycelium on their surface. Non-inoculated and mock-inoculated pods with sterile PDA plugs served as control. Inoculated and control pods were incubated inside a clear plastic box (11 × 11 × 3.5 cm) and over moistened filter paper under 12-h photoperiod at 25 ± 1°C. A surrounding water-soaked halo was visible only on pods inoculated with the fungus 48 h after inoculation (hai). Intense sporulation and necrosis were visible 96 hai. Botrytis spp. was previously detected, through standard blotter test, on seeds of P. sativum in Brazil, but without pathogenicity test nor its transmission through seeds (2,3). To our knowledge, this is the first report of B. fuckeliana causing epidemics on pea pods in Brazil. The high incidence of the disease in a protected environment has the potential to cause significant economic impact due to its damage to the pods, rendering them unmarketable. References: (1) N. L. Glass and G. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) M. A. S. Mendes et al. Fungos em Plantas no Brasil. Embrapa-Cenargen, Brasília, 1998. (3) W. M. Nascimento and S. M. Cícero. Rev. Bras. Sementes 13:5, 1991. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Powdery Mildew Caused by Golovinomyces sp. on Plantago australis in Brazil.

The plantain Plantago australis Lam. (Plantaginaceae) is a herbaceous species native to southern Brazil that is known for the analgesic, antibiotic, and anti-inflammatory properties of its leaf extracts (2). Powdery mildew was observed on wild P. australis plants in the cities of Tapejara, Jari, and Santa Maria (State of Rio Grande do Sul, Brazil) during the summer of 2011. Affected plants were more often observed in shaded areas. Signs included sparse to abundant white powdery masses of conidia and mycelium on pseudo-petioles and leaves, mostly on the adaxial surface. Severely affected plants (≥80% of foliar area affected) had small chlorotic leaves and reduced size compared to healthy ones. Mycelia were superficial and presented nipple-shaped appressoria. Conidiophores were often curved at the base, unbranched, cylindrical, 81 to 125 µm long (average 97.3 ± 14.9 µm) and composed of a cylindrical foot cell 52 to 73 µm long (average 65.4 ± 7.5 µm) and 9 to 14 µm wide (average 11.6 ± 1.5 µm) followed by one to two shorter cells 17 to 29 µm long (average 23.4 ± 3.6 µm). Conidia were produced in chains of up to eight cells, did not contain fibrosin bodies, ranged from ellipsoid-ovoid to subcylindrical, and measured 24 to 35 µm long (average 30.5 ± 3.7 µm) and 12 to 19 µm wide (average 15.8 ± 1.7 µm). Germ tubes were produced apically (reticuloidium type). Chasmothecia were not observed on sampled leaves. Genomic DNA was extracted from conidia, conidiophores, and mycelium and used to amplify the internal transcribed spacer (ITS) (ITS1-5.8s-ITS2) region using the ITS1 and ITS4 primers. The resulting sequence (558 bp) was deposited under accession number JX312220 in GenBank. Searches with the BLASTn algorithm revealed similarity of 100% with Golovinomyces orontii (Castagne) V.P. Heluta 1988 from Veronica arvensis L. (AB077652.1) (3), 99% with G. orontii from Galium spurium L. and Galium aparine L. (AB430818.1 and AB430813.1) (2) and 99% with G. sordidus (L. Junell) V.P. Heluta 1988 from P. lanceolata L. (AB077665.1) (3). Based on morphological characteristics and sequence analysis of the ITS region, the fungus was identified as belonging to Golovinomyces sp. To fulfill Koch's postulates, five cultivated plants of P. australis with four to five expanded leaves were inoculated by dusting conidia (10 to 15 conidia cm-2) on their leaves. Inoculated and non-inoculated control plants were kept in a greenhouse at 27 ± 5°C and relative humidity of 80 ± 15%. Powdery mildew symptoms identical to those of wild plants were observed 8 to 10 days after in inoculated plants. Although G. sordidus was previously reported on P. australis subsp. hirtella in Argentina and on several species of Plantago in others world regions (1), to our knowledge, Golovinomyces sp. has not been previously reported as a pathogen of P. australis in Brazil. Although the economic impact of the disease is limited, the reduction in plant size and leaves affects biomass production used in the extraction of pharmaceutical compounds. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series 11, 2012. (2) G. C. Sousa et al. J. Ethnopharmacol. 90:135, 2004. (3) S. Takamatsu et al. Mycol. Res. 113:117, 2009.

First Report of Powdery Mildew on Flamboyant Tree Caused by Erysiphe quercicola in Brazil.

Flamboyant (Delonix regia) is an ornamental tree that is native to Madagascar and frequently used in gardens and parks worldwide. Powdery mildew was observed on flamboyant plants in the cities of Piracicaba and São Carlos (State of São Paulo, Brazil) during the springs of 2010 and 2011. All sampled plants (~15 plants) were affected by the disease. Affected plants had abundant, white powdery masses of conidia and mycelium on floral buds that is typical of powdery mildew, but these structures were not observed on leaves and petioles. Diseased buds were observed at all developmental stages. The fungus was identified as Erysiphe quercicola on the basis of scanning electron microscopy, light microscopy, and sequence analysis of the internal transcribed spacer (ITS) region. Conidia were produced in short chains of four to five spores on erect conidiophores. Conidiophores were unbranched, cylindrical, 50 to 80 µm long (mean 68.8 ± 10.8 µm), composed of a cylindrical foot cell 25 to 40 µm long (mean 32.2 ± 4.9 µm), and one to two shorter cells. Conidia were ellipsoid-ovoid to subcylindrical, 22 to 37 µm long (mean 30.9 ± 4.4 µm), and 10 to 18 µm wide (mean 15.1 ± 2.8 µm). Germ tubes were produced apically and ended in a lobed appressorium. Colonizing hyphae also had a well-developed lobed appressorium. Chasmothecia were not observed on buds. DNA was extracted from conidia, conidiophores, and mycelium and used to amplify the ITS (ITS1-5.8s-ITS2) region using the ITS1 and ITS4 primers (2) and its sequence (612 nt) was deposited under Accession No. JQ034229 in the GenBank. Searches with the BLASTn algorithm revealed 100% similarity with E. quercicola from oak (Accession Nos. AB292693.1, AB292691.1, and AB292690.1) (1). To fulfill Koch's postulates, 10 detached young floral buds, 0.4 to 0.8 cm in diameter, were inoculated with five to eight conidia collected on floral buds using an eyelash brush. Inoculated buds were placed on moistened filter paper in petri dishes. The negative control consisted of noninoculated young floral buds. Inoculated and noninoculated buds were incubated in a growth chamber at 25°C and a 12-h photoperiod. Powdery mildew structures were observed 6 to 8 days after inoculation. To our knowledge, E. quercicola has not been reported previously as a pathogen of flamboyant tree since there is no record in the Erysipahales database ( http://erysiphales.wsu.edu/ ). Although the economic impact of the disease is limited, its incidence might induce the abortion of floral buds and accelerate the senescence of flowers, thus reducing the aesthetic value of the trees. References: (1) S. Takamatsu et al. Mycol Res. 111:809, 2007. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Root-Knot Disease Caused by Meloidogyne arenaria and M. javanica in Teak in São Paulo State, Brazil.

Teak (Tectona grandis Linn. F.) is one of the most important forest crops in Brazil, occupying areas in different regions, such as Goiás, Mato Grosso, Paraná, and São Paulo states. Teak wood is used for many purposes such as shipbuilding, rolling and plywood, firewood, and charcoal. In May 2011, teak symptomatic feeder root samples, exhibiting inconspicuous, small galls, were collected in the municipality of Piracicaba, São Paulo State, Brazil (22°41'46.90″S, 47°38'36.84″W). Specimens were identified through perineal patterns and esterase phenotypes of 20 adult females (1,2). Perineal patterns and esterase phenotypes were consistent with those described for Meloidogyne arenaria (Neal, 1889) Chitwood, 1949 and M. javanica (Treub, 1885) Chitwood, 1949. Perineal patterns of M. arenaria showed a low dorsal arch, compressed dorsolaterally, with lateral field marked by some forked and broken striae; no punctate markings between anus and tail terminus were observed. Perineal patterns of M. javanica were rounded, with low dorsal arch, striae smooth, lateral field distinct, clearly demarcated from striae by parallel lines. From the esterase electrophoresis we obtained A2 (Rm:1.2;1.3) and J3 (Rm:1.0;1.25;1.4) phenotypes, typical from M. arenaria and M. javanica, respectively. To our knowledge, this is the first report of M. arenaria parasitizing teak roots in Brazil and elsewhere (new host) and the first report of M. javanica infecting teak in the State of São Paulo. Previously, M. javanica was reported to be infecting teak-growing areas in the State of Mato Grosso (3). This finding has a great importance, not only by the inclusion of these parasites in teak pathological scenario, but also for predicting possible damage in plant species used in teak-based intercropping systems. References: (1) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 22:10, 1990. (2) K. M. Hartman and J. N. Sasser. 1985. Page 115 in: An Advanced Treatise on Meloidogyne. Volume II, Methodology. K. R. Barker et al., eds. North Carolina State University Graphics, Raleigh,1985. (3) R. A. Silva et al. Nematol. Bras. 27:261, 2003.

First Report of Tomato chlorosis virus Infecting Tomato Crops in Brazil.

During 2006 and 2007 in the region of Sumaré, state of São Paulo, Brazil, surveys were done on tomato (Solanum lycopersicum L.) virus diseases in three open field-grown crops. The data revealed low incidence (0.25 to 3.42%) of randomly distributed plants exhibiting interveinal chlorosis and some necrosis on the basal leaves. Symptoms were only observed on old fruit-bearing plants. Preliminary analysis of thin sections of symptomatic leaves from one plant by transmission electron microscopy revealed the presence of aggregates of thin, flexible, and elongated particles in some phloem vessels, suggesting infection with a member of the genus Crinivirus, family Closteroviridae. Total RNA was extracted separately from leaves of 10 symptomatic plants and used for one-step reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (1). The RT-PCR product was subsequently tested by nested-PCR for single detection of TICV and ToCV using primer pairs TIC-3/TIC-4 and ToC-5/ToC-6, respectively (1). Only one fragment of approximately 463 bp was amplified from 7 of the 10 plants with the primer pair specific for ToCV. No amplification was obtained with the primers specific for TICV. Two amplicons of 463 bp were purified and directly sequenced in both directions. Sequence comparisons of the 463-bp consensus sequence (GenBank Accession No. EU868927) revealed 99% identity with the reported sequence of ToCV from the United States (GenBank Accession No. AY903448) (3). Virus-free adults of Bemisia tabaci biotype B confined on symptomatic tomato leaves for a 24-h acquisition access period were able to transmit the virus to healthy tomato plants, which reproduced the original symptoms on the bottom leaves 65 days after inoculation under greenhouse conditions. Infection from transmission was confirmed by RT-PCR using the HS-11/HS-12 primer pair. In addition to B. tabaci biotype B, the greenhouse whitefly, Trialeurodes vaporariorum, has also been reported as a vector of ToCV, although it is less efficient than the B. tabaci biotype B in transmission of this virus (4). T. vaporariorum, which was previously considered limited to greenhouses, was recently reported in tomato and green bean (Phaseolus vulgaris L.) crops under field conditions in São Paulo State (2). Therefore, it might also contribute to the spread of ToCV in tomato crops in São Paulo. To our knowledge, this is the first report of ToCV in Brazil and South America. References: (1) C. I. Dovas et al. Plant Dis.86:1345, 2002. (2) A. L. Lourenção et al. Neotrop. Entomol. 37:89, 2008. (3) W. M. Wintermantel et al. Arch. Virol. 15:2287, 2005. (4) W. M. Wintermantel and G. C. Wisler. Plant Dis. 90:814, 2006.

The anthracnose resistance locus Co-4 of common bean is located on chromosome 3 and contains putative disease resistance-related genes.

The broadest based resistance to anthracnose of common bean ( Phaseolus vulgaris L.) is conferred by the Co-4 locus. We sequenced a bacterial artificial chromosome clone harboring part of the Co-4 locus of the bean genotype Sprite and assembled a single contig of 106.5 kb for functional annotation. This region contained five copies of the COK-4 gene that encodes for a serine threonine kinase protein previously mapped to the Co-4 locus and 19 novel genes with no similarity to any previously identified genes of common bean. Several putative genes of the Co-4 locus seemed to be expressed as they matched perfectly with bean expressed sequence tags. The expression of the COK-4 genes was assessed by reverse transcription (RT)-PCR, and a single 850-bp cDNA fragment was sequenced and compared with the genomic sequences of the COK-4 homologs. Although the COK-4 cDNA was isolated from a different bean cultivar, it showed high similarity (95%) to the exons of genes BA17 and BA21, suggesting that they were expressed. In a phylogenetic tree including all currently available Pto-like sequences from Phaseolus species, the COK-4 homologs formed a single cluster with the Pto gene, whereas two sequences from P. coccineus and all sequences of P. vulgaris formed two closely related clusters. The Co-4 locus was physically mapped to the short arm of bean chromosome 3, which corresponds to linkage group B8. This study represents a first step in gaining an understanding of the genomic organization of an anthracnose resistance locus of common bean and provides molecular data for comparative analysis with other plant species.

Genome features of Leptospira interrogans serovar Copenhageni.

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Genome features of Leptospira interrogans serovar Copenhageni

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Occurrence of Podosphaera xanthii Race 2 on Cucumis melo in Brazil.

Powdery mildew is an important disease of melons (Cucumis melo L.) cultivated in greenhouses in Brazil. Currently, there are 5 races of Podosphaera xanthii (formerly known as Sphaerotheca fuliginea) and 2 races of Golovinomyces cichoracearum (formerly known as Erysiphe cichoracearum) described on melons worldwide, but only race 1 of P. xanthii has been reported in Brazil (1). However, typical whitish powdery fungal growth was observed on an experimental hybrid yellow melon resistant to race 1 of P. xanthii during the summer of 2000 in a greenhouse in Bragança Paulista, State of São Paulo. Conidia collected from diseased leaves were spread onto 0.5% water agar medium and maintained at 22°C for 24 h with 12 h of light and 12 h of darkness. Most of the germinated conidia displayed fibrosin inclusion bodies when observed in a solution of 3% potassium hydroxide (KOH), and approximately 1 of 50 also displayed forked germ tubes. These features allowed us to identify P. xanthii as the causal agent. Conidia raised on the susceptible yellow melon 'Amarelo CAC' were used to inoculate cotyledons of the differential melon lines (2) 'Hale's Best Jumbo' (susceptible to races 1, 2, and 3 of P. xanthii), 'PMR-45' (resistant to race 1 and susceptible to races 2 and 3), and 'PMR-6' (resistant to races 1 and 2 and susceptible to race 3). Inoculations were performed on 10 plants of each differential line and replicated four times. The presence or absence of symptoms was evaluated 18 days after inoculation. 'Hale's Best Jumbo' and 'PMR-45' were rated as susceptible while 'PMR-6' was rated as resistant, thus indicating the presence of race 2 of P. xanthii in Brazil. During field surveys from 2001 to 2003, this race was found on squash (Cucurbita moschata), summer squash (C. pepo), and melons in São Paulo. References: (1) F. J. B. Reifschneider et al. Plant Dis. 69:1069, 1985. (2) C. E. Thomas et al. Cucurbit Genet. Coop. 7:126, 1984.

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.

Comparative genomic analysis of plant-associated bacteria.

This review deals with a comparative analysis of seven genome sequences from plant-associated bacteria. These are the genomes of Agrobacterium tumefaciens, Mesorhizobium loti, Sinorhizobium meliloti, Xanthomonas campestris pv campestris, Xanthomonas axonopodis pv citri, Xylella fastidiosa, and Ralstonia solanacearum. Genome structure and the metabolism pathways available highlight the compromise between the genome size and lifestyle. Despite the recognized importance of the type III secretion system in controlling host compatibility, its presence is not universal in all necrogenic pathogens. Hemolysins, hemagglutinins, and some adhesins, previously reported only for mammalian pathogens, are present in most organisms discussed. Different numbers and combinations of cell wall degrading enzymes and genes to overcome the oxidative burst generally induced by the plant host are characterized in these genomes. A total of 19 genes not involved in housekeeping functions were found common to all these bacteria.

Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Xanthomonas albilineans Haplotype B Responsible for a Recent Sugarcane Leaf Scald Disease Outbreak in Cuba.

Over the past 3 years, the incidence of sugarcane leaf scald disease (LSD) caused by Xanthomonas albilineans has increased at alarming rates in some Caribbean countries. LSD was in latent phase since 1978, when the disease was reported in Cuba, until February 1998 when typical symptoms were observed in germ plasm collections and in some commercial plantings. More than 150 bacterial isolates from different sugarcane varieties and from different localities were isolated on Wilbrink agar medium and characterized. All isolates had shown similar cultural and biochemical patterns. However, serological differences between isolates from the recent outbreak and the ones obtained prior to 1998 were detected by indirect ELISA testing. Differences between Cuban isolates obtained prior to 1998 and those from the recent outbreak were confirmed by analysis of repetitive DNA sequences dispersed throughout the genome. According to the pattern obtained, the newer isolates were similar to reference strains classified as haplotype B by pulsed field gel electrophoresis (1). It is concluded that the recent outbreak of LSD was caused by a strain different than the ones previously detected in Cuba. Reference: (1) M. J. Davis et al. Phytopathology 87:316, 1997.