Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads

Abstract Background Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. Results Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. Conclusions This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

Detección de Xanthomonas cucurbitae (Bryan) Vauterin et al. afectando frutos de zapallo coreano en Mendoza y San Juan, Argentina

Durante las campañas 2007/08 y 2008/09, en explotaciones comerciales de zapallo coreano (Cucurbita moschata Duch) de las zonas hortícolas de Mendoza y San Juan, se observaron sobre los frutos numerosas lesiones circulares, de 3 a 6 mm de diámetro, de aspecto húmedo, algo deprimidas hacia el centro, con un exudado gomoso color ámbar. Cuando las lesiones superficiales se unían, se desarrollaba una podredumbre gelatinosa hacia el interior de los tejidos, que podía profundizar hasta la cavidad seminal. El resultado final de la afección era una podredumbre seca y costrosa, que abarcaba gran parte del fruto, inutilizándolo para ser comercializado. En follaje también se observaron síntomas de la enfermedad, como manchas cloróticas angulares de 2 a 3 mm de lado, que en ocasiones se fusionaban. El objetivo del presente trabajo fue determinar la etiología de la enfermedad. Para ello, se tomaron y analizaron muestras de frutos y hojas afectadas, durante ambas temporadas. Se concluyó que la sintomatología es causada por una bacteria, que de acuerdo con los estudios morfobioquímicos y patogénicos es Xanthomonas cucurbitae (Bryan) Vauterin et al. Esta investigación constituye la primera cita de este patógeno afectando frutos en Argentina.

The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin. The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases. The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors. Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor. These data strongly suggest that AlbD is an albicidin hydrolase. The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35°C. Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane. The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp

Wolbachia are bacteria that live in the cells of various invertebrate species to which they cause a wide range of effects on physiology and reproduction. We investigated the effect of Wolbachia infection in the parasitic wasp, Asobara tabida Nees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected by Wolbachia. The removal of Wolbachia by antibiotic treatment had a totally unexpected effect—aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treated Asobara citri, a closely related species that does not harbor Wolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents) are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production and Wolbachia density in females. We conclude that Wolbachia is necessary for oogenesis in these A. tabida strains, and this association would seem to be the first example of a transition from facultative to obligatory symbiosis in arthropod–Wolbachia associations.

Is the potential of Coccidoxenoides perminutus, a mealybug parasitoid, limited by climatic or nutritional factors?

The encyrtid Coccidoxenoides perminutus is a widely distributed parasitoid of citrus mealybug (Planococcus citri). Worldwide, it has been implicated in successful biocontrol in only a few widely separated localities. C perminutus contributes little to control P. citri in field situations in south-east Queensland, Australia, but invades insectary cultures and reduces mealybug populations considerably under these controlled conditions. This discrepancy between poor field performance and good performance under controlled conditions was investigated to establish whether climatic factors inhibit the field performance of this species in the biological control of P. citri. Subsequent laboratory examination of the influence of varied humidities and temperatures on the activity levels and survival of C perminutus revealed a low tolerance for high saturation deficits (i.e., low % RH at high T degreesC) with reduced reproductive output. The influence of different food sources on adult survival and reproduction was also quantified, to establish if the adverse effects of climate could be overcome by supplementing adult diet. Neither honeydew from their mealybug hosts nor nectar from Alphitonia flowers significantly enhanced parasitoid survival. A subsequent test of five nectar species revealed a significant difference in their influence on C. perminutus survival and reproduction, with only Alpinia zerumbet proving to be as suitable as honey. The floral species that proved suitable in the laboratory need to be checked for their attractiveness to C perminutus in the field and for their ability to enhance the survival and reproductive output of parasitoids. This information suggests that the prevailing dry conditions in south-east Queensland citrus-growing areas apparently impede successful biological control of P. citri by C perminutus, but possibilities are available for habitat manipulation (by providing suitable nectar sources for adult parasitoids) to conserve and enhance C perminutus activity in the field. (C) 2004 Elsevier Inc. All rights reserved.

Life history parameters and biocontrol potential of the mealybug parasitoid Coccidoxenoides peregrinus (Timberlake) (Hymenoptera: Encyrtidae): Asexuality, fecundity and ovipositional patterns

Properties relevant to the ovipositional activity and lifetime productivity of Coccidoxenoides peregrinus (Timberlake) were assessed in the laboratory, to determine the potential of this species as a biocontrol agent against the citrus mealybug, Planococcus citri (Risso). In general, this species has not performed well in orchards, except for a few localities on different continents. The mode of reproduction of C peregrinus is almost entirely thelytokous, with males produced sporadically and at low frequency. The females have both pro-ovigenic and synovigenic traits, which raises questions of the utility of this distinction. The females have a high reproductive potential with 10-20 eggs per day available within the first two days (after a short (12 h) pre-oviposition period), and 80-150 eggs per day thereafter until death at about eight days. Mean lifetime fecundity was 239.2 +/- 34.3 eggs. C peregrinus oviposits across a range of P. citri instars, but productivity relies predominantly on second instar hosts. Second stage (N2) hosts received most eggs in choice (about 52%) and no-choice (about 50%) tests. Most eggs deposited into N2 hosts (82%) reached adult stage whereas only a few of those deposited into N1 and N3 (about 5% each) developed successfully. The haemolymph of parasitised reproductive mealybugs contained granular structures and no parasitoid eggs were found 24 h after exposure to ovipositing wasps. Also, no wasps emerged from parasitised adult hosts that were kept alive. Parasitoid eggs deposited into adult hosts were presumed encapsulated and destroyed, as control mealybugs (not exposed to female wasps) had no granular structures in their haemolymph. Wasps exposed to an abundance of hosts soon started ovipositing, but only for a relatively short time each day (about 2.5 h out of a 7 h exposure). They stopped ovipositing despite eggs judged to be mature in their ovaries. The reproductive output of C peregrinus is discussed in relation to the ecological factors that could influence this output, and the implications for biocontrol are discussed. (C) 2003 Elsevier Inc. All rights reserved.

Why is Coccidoxenoides perminutus, a mealybug parasitoid, ineffective as a biocontrol agent - Inaccurate measures of parasitism or low adult survival?

Coccidoxenoides perminutus achieves only low levels of parasitism of its host Planococcus citri in southeast Queensland citrus. Two possible causes were investigated. Adult survival under natural conditions was assessed to determine whether providing adult food sources could enhance survival. Behavioural changes of hosts, induced by C perminutus parasitism, was also investigated to establish if parasitised P. citri move from their feeding site to seek protected shelters some distance away and are thus not accounted for in field assessments of parasitism rates. Unparasitised mealybugs placed in the field for two periods were retrieved before the effects of parasitism were manifested and parasitism rates were still low (0.3% at 5 days and 1.2% at 10 days). Levels of locomotion of P. citri exposed to C perminutus were compared with those of unexposed ones. Parasitised mealybugs, regardless of instar, undergo behavioural changes. In comparison to unparasitised controls, the mealybugs become highly active 7-14 days after exposure to wasps. All parasitised mealybugs undergo physical changes, their body becomes cylindrical, their legs go so rigid that the mealybugs become immobile, and this signifies the typical mummy appearance. All mealybugs that became mummies eventually fell from the host lemon fruit because of impaired locomotion and were caught on sticky traps that had been placed beneath the lemons. Consequently, their final site of mummification was not established. C perminutus adults provided with nectar or honey survived longer (about 5 days) in the field than those without food (about a day). Nectar from two plant species, Alpinia zerumbet and Datura candida, proved to be good sources of food for the adult wasps, and were comparable in quality to honey. The low level of parasitism achieved by C perminutus in southeast Queensland citrus thus appears to be a consequence of the short adult life and the negative effects of a harsh environment. Provision of a suitable food source (e.g., nectar) may well enhance levels of parasitism in the field. (c) 2005 Elsevier Inc. All rights reserved.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

AnnoTALE: Bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present 'AnnoTALE', a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities. © 2016, Nature Publishing Group. All rights reserved.

Efeito de diferentes concentrações de andiroba na inibição do crescimento in vitro da bactéria Xanthomonas axonopodis pv. passiflorae.

Na composição química das plantas medicinais, algumas substâncias podem atuar como ativadoras do sistema defensor da planta hospedeira ou contra os patógenos fúngicos. Na constituição química. As amostras de óleo de andiroba utilizadas nos testes foram do Horto de Plantas Medicinais da Embrapa Amazônia Oriental, Belém- PA. Para o crescimento, os patógenos foram cultivados em meio de cultura MB1 sintético. A amostra utilizada na verificação da inibição fitopatogênica foi óleo puro de andiroba em três concentrações de 1%, 2% e 3% para a bactéria. O delineamento experimental foi inteiramente casualizado, sete tratamentos (uma espécie de bactéria X três concentrações do óleo de andiroba) e cinco repetições. A análise estatística foi realizada comparando as medidas pelo teste de Tukey a 5% de probabilidade utilizando o programa estatístico SISVAR. O óleo de andiroba apresentou efeito significativo na inibição do crescimento da bactéria em todas as concentrações utilizadas, onde a maior concentração do óleo de andiroba mostrou-se mais eficiente na inibição do crescimento da bactéria, em relação à testemunha.

Ocorrência de joaninha Azya luteipes 1850 Mulsant (Coleoptera: Coccinellidae), agente de controle biológico de cochonilhas do cafeeiro, em Porto Velho, Rondônia.

Publicação que reúne informações sobre a ocorrência de joaninha Azya luteipes em plantações de café Conilon, no município de Porto Velho, Rondônia.