Genetic Diversity and a PCR-Based Method for Xanthomonas axonopodis Detection in Passion Fruit

Xanthomonas axonopodis pv. passiflorae causes bacterial spot in passion fruit. It attacks the purple and yellow passion fruit as well as the sweet passion fruit. The diversity of 87 isolates of pv. passiflorae collected from across 22 fruit orchards in Brazil was evaluated using molecular profiles and statistical procedures, including an unweighted pair-group method with arithmetical averages-based dendrogram, analysis of molecular variance (AMOVA), and an assigning test that provides information on genetic structure at the population level. Isolates from another eight pathovars were included in the molecular analyses and all were shown to have a distinct repetitive sequence-based polymerase chain reaction profile. Amplified fragment length polymorphism technique revealed considerable diversity among isolates of pv. passiflorae, and AMOVA showed that most of the variance (49.4%) was due to differences between localities. Cluster analysis revealed that most genotypic clusters were homogeneous and that variance was associated primarily with geographic origin. The disease adversely affects fruit production and may kill infected plants. A method for rapid diagnosis of the pathogen, even before the disease symptoms become evident, has value for producers. Here, a set of primers (Xapas) was designed by exploiting a single-nucleotide polymorphism between the sequences of the intergenic 16S-23S rRNA spacer region of the pathovars. Xapas was shown to effectively detect all pv. passiflorae isolates and is recommended for disease diagnosis in passion fruit orchards.

Tree performance and fruit yield and quality of `Okitsu` Satsuma mandarin grafted on 12 rootstocks

The citriculture in Brazil, as well as in other important regions in the world, is based on very few mandarin cultivars. This fact leads to a short harvest period and higher prices for off-season fruit. The `Okitsu` Satsuma (Citrus unshiu Marc.) is among the earliest ripening mandarin cultivars and it is considered to be tolerant to, citrus canker (Xanthomonas citri subsp. citri Schaad et al.) and to citrus variegated chlorosis (Xylella fastidiosa Wells et al.). Despite having regular fruit quality under hot climate conditions, the early fruit maturation and absence of seeds of `Okitsu` fruits are well suited for the local market in the summer(December through March), when the availability of citrus fruits for fresh consumption is limited. Yet, only a few studies have been conducted in Brazil on rootstocks for `Okitsu`. Consequently, a field trial was carried out in Bebeclouro, Sao Paulo State, to evaluate the horticultural performance of `Okitsu` Satsuma mandarin budded onto 12 rootstocks: the citrandarin `Changsha` mandarin (Citrus reticulata Blanco) x Poncirus trifoliata `English Small`: the hybrid Rangpur lime (Citrus limonia Osbeck) x `Swingle` citrumelo (P. trifoliata (L.) Raf. x Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L) Raf)`Rubidoux`,`FCAV` and `Flying Dragon`(P. trifoliata var. monstrosa); the mandarins `Sun Chu Sha Kat`(C. reticulata Blanco) and `Sunki`(Citrus sunki (Hayata) Hort. ex. Tanaka); the Rangpur limes (C. limonia Osbeck) `Cravo Limeira` and `Cravo FCAV`;`Carrizo` citrange (Citrus sinensis x P. trifoliata), `Swingle` citrumelo (P. trifoliata x C. paradisi), and `Orlando` tangelo (C. paradisi x Citrus tangerina cv. `Dancy`). The experimental grove was planted in 2001, using a 6 m x 3 m spacing, in a randomized block design. No supplementary irrigation was applied. Fruit yield, canopy volume, and fruit quality were assessed for each rootstock. A cluster multivariate analysis identified three different rootstock pairs with similar effects on plant growth, yield and fruit quality of `Okitsu` mandarin. The `Flying Dragon `trifoliate had a unique effect over the `Okitsu` trees performance, inducing lower canopy volume and higher yield efficiency and fruit quality, and might be suitable for high-density plantings. The `Cravo Limeira` and `Cravo FCAV` Rangpur limes induced early-ripening of fruits, with low fruit quality. `Sun Chu Sha Kat` and `Sunki` mandarins and the `Orlando` tangelo conferred lower yield efficiency and less content of soluble solids for the latter rootstock. (C) 2009 Elsevier B.V. All rights reserved.

Horticultural performance of `Folha Murcha` sweet orange onto twelve rootstocks

Despite its outstanding position, the Brazilian citriculture is established on a very limited pool of varieties that limits its expansion and restricts the fruit availability throughout the year. This situation determines the urgent necessity of developing alternative scion and rootstock cultivars, with good performance under local conditions. `Folha Murcha` sweet orange (Citrus sinensis (L.) Osbeck) is a late-harvest cultivar, suitable both for the juice processing industry and the fresh fruit market, being described as tolerant to citrus canker (Xanthomonas citri subsp. citri Schaad et al.), and less affected by citrus variegated chlorosis (Xylella fastidiosa Wells et al.). A study was conducted in Bebedouro, Sao Paulo State, Brazil, to evaluate the horticultural performance of `Folha Murcha` sweet orange budded onto 12 rootstocks: the citrandarin `Changsha` mandarin (Citrus reticulata Blanco) x Poncirus trifoliata `English Small`: the hybrid `Rangpur` lime (Citrus limonia Osbeck) x `Swingle` citrumelo (P. trifoliata (L.) Raf x Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L.) Raf.)`Rubidoux`, `FCAV`, and `Flying Dragon` (P. trifoliata var. monstrosa); the `Sun Chu Sha Kat` mandarin (C. reticulata Blanco); the `Sunki` mandarin (Citrus sunki (Hayata) Hart. ex. Tanaka); the `Rangpur` limes (C. limonia Osbeck) `Cravo Limeira` and `Cravo FCAV`; `Carrizo` citrange (C. sinensis x P. trifoliata), `Swingle` citrumelo (P. trifoliata x C. paradisi), and `Orlando` tangelo (C. paradisi x Citrus tangerina cv. `Dancy`). The experimental grove was planted in 2001, using a 7 m x 4 m spacing, in a randomized block design, with five replications and two plants per plot. No supplementary irrigation was applied. Fruit yield, canopy volume, tree tolerance to drought and to citrus variegated chlorosis, and fruit quality were assessed for each rootstock. Trees grafted onto the `Flying Dragon` trifoliate were smaller in size, but had largest yield efficiency when compared to those grafted onto other rootstocks. Lower alternate bearing index was observed on trees budded onto `Cravo FCAV` `Rangpur` lime. Both `Rangpur` lime rootstocks and the `Sunki` mandarin induced higher tree tolerance to drought. The `Flying Dragon` trifoliate induced better fruit quality and higher tolerance to citrus variegated chlorosis (CVC) to `Folha Murcha` trees. A cluster multivariate analysis identified three groups of rootstocks with similar effects on `Folha Murcha` tree performance. Among the 12 evaluated rootstocks, the `Flying Dragon` trifoliate has a unique effect on plant growth, tolerance to drought and CVC, fruit yield and fruit quality of `Folha Murcha` trees, and may be better suited for high-density plantings. (C) 2011 Elsevier B.V. All rights reserved.

Genetic diversity and plant-growth related features of Burkholderia spp. from sugarcane roots

Brazil is the largest sugarcane producer in the world, mainly due to the development of different management strategies. Recently, microbial-plant related studies revealed that bacterial isolates belonging to the genus Burkholderia are mainly associated with this plant and are responsible for a range of physiological activity. In this study, we properly evaluate the physiological activity and genetic diversity of endophytic and rhizospheric Burkholderia spp. isolates from sugarcane roots grown in the field in Brazil. In total, 39 isolates previously identified as Burkholderia spp. were firstly evaluated for the capability to fix nitrogen, produce siderophores, solubilise inorganic phosphates, produce indole-acetic acid and inhibit sugarcane phytopathogens in vitro. These results revealed that all isolates present at least two positive evaluated activities. Furthermore, a phylogenetic study was carried out using 16S rRNA and gyrB genes revealing that most of the isolates were affiliated with the Burkholderia cepacia complex. Hence, a clear separation given by endophytic or rhizospheric niche occupation was not observed. These results presented an overview about Burkholderia spp. isolates from sugarcane roots and supply information about the physiological activity and genetic diversity of this genus, given direction for further studies related to achieve more sustainable cultivation of sugarcane.

Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

Bacterial community in the rhizosphere and rhizoplane of wild type and transgenic eucalyptus

The rhizosphere is a niche exploited by a wide variety of bacteria. The expression of heterologous genes by plants might become a factor affecting the structure of bacterial communities in the rhizosphere. In a greenhouse experiment, the bacterial community associated to transgenic eucalyptus, carrying the Lhcb1-2 genes from pea (responsible for a higher photosynthetic capacity), was evaluated. The culturable bacterial community associated to transgenic and wild type plants were not different in density, and the Amplified Ribosomal DNA Restriction Analysis (ARDRA) typing of 124 strains revealed dominant ribotypes representing the bacterial orders Burkholderiales, Rhizobiales, and Actinomycetales, the families Xanthomonadaceae, and Bacillaceae, and the genus Mycobacterium. Principal Component Analysis based on the fingerprints obtained by culture-independent Denaturing Gradient Gel Electrophoresis analysis revealed that Alphaproteobacteria, Betaproteobacteria and Actinobacteria communities responded differently to plant genotypes. Similar effects for the cultivation of transgenic eucalyptus to those observed when two genotype-distinct wild type plants are compared.

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Long-Chain Acyl-Homoserine Lactones from Methylobacterium mesophilicum: Synthesis and Absolute Configuration

The acyl-homoserine lactones (acyl-HSLs) produced by Methylobacterium mesophilicum isolated from orange trees infected with the citrus variegated chlorosis (CVC) disease have been studied, revealing the occurrence of six long-chain acyl-HSLs, i.e., the saturated homologues (S)-N-dodecanoyl (1) and (S)-N-tetradecanoyl-HSL (5), the uncommon odd-chain N-tridecanoyl-HSL (3), the new natural product (S)-N-(2E)-dodecenoyl-HSL (2), and the rare unsaturated homologues (S)-N-(7Z)-tetradecenoyl (4) and (S)-N-(2E,7Z)-tetradecadienyl-HSL (6). The absolute configurations of all HSLs were determined as 3S. Compounds 2 and 6 were synthesized for the first time. Antimicrobial assays with synthetic acyl-HSLs against Gram-positive bacterial endophytes co-isolated with M. mesophilicum from CVC-infected trees revealed low or no antibacterial activity.

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.

Espécies de cigarrinhas (Hemiptera, Cicadellidae, Cicadellinae) em pomares de citros da região de Bebedouro, SP, com descrição de uma espécie nova de Acrogonia Stål

Sharpshooter leafhoppers (Hemiptera, Cicadellidae, Cicadellinae) in citrus groves of the area of Bebedouro, SP, with description of a new species of Acrogonia Stål. Sixteen species of Cicadellidae, subfamily Cicadellinae, were collected and identified: 11 of the tribe Cicadellini and five of the tribe Proconiini. The ten most common species, together with a new species, are characterized based on external morphology and male genitalia: Acrogonia citrina Marucci & Cavichioli sp. nov.; Bucephalogonia xanthophis (Berg, 1879); Dilobopterus costalimai Young, 1977; Ferrariana trivittata (Signoret, 1854); Hortensia similis (Walker, 1851); Macugonalia cavifrons (Stål, 1862); M. leucomelas (Walker, 1851); Oncometopia facialis (Signoret, 1854); Plesiommata corniculata Young, 1977 and P. mollicella (Fowler, 1900). The leafhopper samples were collected in citrus groves and in adjacent habitats (woods, swamp, cultivable plain, spontaneous vegetation of the grove, coffee crop and sugarcane) at the counties of Bebedouro and Monte Azul Paulista and in the district of Turvínia. The collecting methods were: insect net, yellow sticky cards, portable suction trap and Malaise trap. Identification keys were elaborated for the 16 species. There is a great diversity of Cicadellinae species in the citric groves. In the three areas sampled, it was collected a larger number of species of the tribe Cicadellini than of the tribe Proconiini. In the Bebedouro area, the vector species of Xylella fastidiosa, A. citrina sp. nov., B. xanthophis, D. costalimai and O. facialis, were collected in the citrus groves and in all adjacent habitats.

Caracterização da comunidade bacteriana endofítica de citros por isolamento, PCR específico e DGGE

O objetivo deste trabalho foi caracterizar a comunidade bacteriana endofítica de plantas assintomáticas (escapes) e afetadas pela clorose variegada dos citros (CVC) por meio de isolamento em meio de cultura, técnica de gradiente desnaturante em gel de eletroforese (DGGE) e detecção de Methylobacterium mesophilicum e Xyllela fastidiosa por meio de PCR específico, para estudar esta comunidade e sua relação com a ocorrência da CVC. A análise da comunidade bacteriana via DGGE permitiu a detecção de X. fastidiosa, bem como Klebsiella sp. e Acinetobacter sp. como endófitos de citros. Foram observados também Curtobacterium sp., Pseudomonas sp., Enterobacter sp. e Bacillus spp. Utilizando primers específicos, Methylobacterium mesophilicum e X. fastidiosa também foram observadas, reforçando hipóteses de que estas bactérias podem estar interagindo no interior da planta hospedeira.

Fluxo de seiva e fotossíntese em laranjeira 'Natal' com clorose variegada dos citros

O objetivo deste trabalho foi avaliar os efeitos da clorose variegada dos citros (CVC), no fluxo de seiva, trocas gasosas e atividade fotoquímica em laranjeira 'Natal', com e sem CVC, em condição de campo. O curso diário do fluxo de seiva, potencial da água na folha, assimilação de CO2, transpiração, condutância estomática e eficiência quântica máxima e efetiva do fotossistema II foram avaliados. O delineamento experimental foi em blocos ao acaso com cinco repetições. O fluxo de seiva foi 1,9 vez superior nas plantas sadias em relação às doentes. Em plantas doentes ocorreu queda de 43, 28 e 33% na assimilação de CO2, condutância estomática e transpiração, respectivamente. As plantas com CVC apresentaram fotoinibição dinâmica. Uma vez que a eficiência quântica efetiva apresentou um padrão de resposta semelhante, durante o dia, em ambos os tratamentos, o efeito protetor da fotorrespiração no aparato fotoquímico em plantas com CVC é discutido. As quedas de assimilação de CO2, transpiração e de fluxo de seiva, nas plantas com CVC, foram decorrentes do menor valor da condutância estomática, possivelmente causado pela colonização dos vasos do xilema pela Xylella fastidiosa.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Bacterial variations on the methionine salvage pathway.

BACKGROUND: The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism. RESULTS: This work uses genome in silico analysis to propose methionine salvage pathways for Klebsiella pneumoniae, Leptospira interrogans, Thermoanaerobacter tengcongensis and Xylella fastidiosa. Experiments performed with mutants of B. subtilis and Pseudomonas aeruginosa substantiate the hypotheses proposed. The enzymes that catalyze the reactions are recruited from a variety of origins. The first, ubiquitous, enzyme of the pathway, MtnA (methylthioribose-1-phosphate isomerase), belongs to a family of proteins related to eukaryotic intiation factor 2B alpha. mtnB codes for a methylthioribulose-1-phosphate dehydratase. Two reactions follow, that of an enolase and that of a phosphatase. While in B. subtilis this is performed by two distinct polypeptides, in the other organisms analyzed here an enolase-phosphatase yields 1,2-dihydroxy-3-keto-5-methylthiopentene. In the presence of dioxygen an aci-reductone dioxygenase yields the immediate precursor of methionine, ketomethylthiobutyrate. Under some conditions this enzyme produces carbon monoxide in B. subtilis, suggesting a route for a new gaseous mediator in bacteria. Ketomethylthiobutyrate is finally transaminated by an aminotransferase that exists usually as a broad specificity enzyme (often able to transaminate aromatic aminoacid keto-acid precursors or histidinol-phosphate). CONCLUSION: A functional methionine salvage pathway was experimentally demonstrated, for the first time, in P. aeruginosa. Apparently, methionine salvage pathways are frequent in Bacteria (and in Eukarya), with recruitment of different polypeptides to perform the needed reactions (an ancestor of a translation initiation factor and RuBisCO, as an enolase, in some Firmicutes). Many are highly dependent on the presence of oxygen, suggesting that the ecological niche may play an important role for the existence and/or metabolic steps of the pathway, even in phylogenetically related bacteria. Further work is needed to uncover the corresponding steps when dioxygen is scarce or absent (this is important to explore the presence of the pathway in Archaea). The thermophile T. tengcongensis, that thrives in the absence of oxygen, appears to possess the pathway. It will be an interesting link to uncover the missing reactions in anaerobic environments.