Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 689
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Syst Appl Microbiol ; 43(1): 126046, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31818496

RESUMO

The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus Agrobacterium contained the non-pathogenic species Agrobacterium radiobacter and the plant pathogenic species Agrobacterium tumefaciens and Agrobacterium rhizogenes. At the end of the past century two new pathogenic species, Agrobacterium rubi and Agrobacterium vitis, were added to the genus. Already in the present century these species plus Agrobacterium larrymoorei were reclassified into genus Rhizobium. This reclassification was controversial and for a time both genus names were used when new species were described. Few years ago, after a taxonomic revision based on genomic data, the old species A. rhizogenes was maintained in the genus Rhizobium, the old species A. vitis was transferred to the genus Allorhizobium and several Rhizobium species were transferred to the genus Agrobacterium, which currently contains 14 species including the old species A. radiobacter, A. tumefaciens, A. rubi and A. larrymoorei. Most of these species are able to produce tumours in different plants, nevertheless the genus Agrobacterium also encompasses non-pathogenic species, one species able to nodulate legumes and one human pathogenic species. Taking into account that the species affiliations to five Agrobacterium genomospecies have not been determined yet, an increase in the number of species within this genus is expected in the near future.


Assuntos
Agrobacterium/classificação , Filogenia , Agrobacterium/genética , DNA Bacteriano/genética , Genes Bacterianos/genética , Genes Essenciais/genética , Genoma Bacteriano/genética , Humanos , Rhizobiaceae/classificação , Rhizobiaceae/genética , Rhizobium/classificação , Rhizobium/genética
2.
BMC Genomics ; 20(1): 643, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405380

RESUMO

BACKGROUND: Ensifer alkalisoli YIC4027, a recently characterized nitrogen-fixing bacterium of the genus Ensifer, has been isolated from root nodules of the host plant Sesbania cannabina. This plant is widely used as green manure and for soil remediation. E. alkalisoli YIC4027 can grow in saline-alkaline soils and is a narrow-host-range strain that establishes a symbiotic relationship with S. cannabina. The complete genome of this strain was sequenced to better understand the genetic basis of host specificity and adaptation to saline-alkaline soils. RESULTS: E. alkalisoli YIC4027 was found to possess a 6.1-Mb genome consisting of three circular replicons: one chromosome (3.7 Mb), a chromid (1.9 Mb) and a plasmid (0.46 Mb). Genome comparisons showed that strain YIC4027 is phylogenetically related to broad-host-range Ensifer fredii strains. Synteny analysis revealed a strong collinearity between chromosomes of E. alkalisoli YIC4027 and those of the E. fredii NGR234 (3.9 Mb), HH103 (4.3 Mb) and USDA257 (6.48 Mb) strains. Notable differences were found for genes required for biosynthesis of nodulation factors and protein secretion systems, suggesting a role of these genes in host-specific nodulation. In addition, the genome analysis led to the identification of YIC4027 genes that are presumably related to adaptation to saline-alkaline soils, rhizosphere colonization and nodulation competitiveness. Analysis of chemotaxis cluster genes and nodulation tests with constructed che gene mutants indicated a role of chemotaxis and flagella-mediated motility in the symbiotic association between YIC4027 and S. cannabina. CONCLUSIONS: This study provides a basis for a better understanding of host specific nodulation and of adaptation to a saline-alkaline rhizosphere. This information offers the perspective to prepare optimal E. alkalisoli inocula for agriculture use and soil remediation.


Assuntos
Adaptação Fisiológica/genética , Meio Ambiente , Genômica , Especificidade de Hospedeiro , Rhizobiaceae/genética , Rhizobiaceae/fisiologia , Genes Bacterianos/genética , Polissacarídeos Bacterianos/biossíntese , Rhizobiaceae/metabolismo , Rizosfera , Solo/química
3.
J Appl Microbiol ; 127(4): 1172-1183, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31329353

RESUMO

AIMS: This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set. METHODS AND RESULTS: The RAPD primer, OPA4, amplified a specific fragment of c. 400 bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330 bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5 pg µl-1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas-specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas simultaneously in root tissues of citrus plants. CONCLUSIONS: A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots. SIGNIFICANCE AND IMPACT OF THE STUDY: A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.


Assuntos
Citrus/microbiologia , Marcadores Genéticos/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Phytophthora/genética , Rhizobiaceae/genética , DNA/análise , DNA/genética , Phytophthora/isolamento & purificação , Rhizobiaceae/isolamento & purificação
4.
BMC Plant Biol ; 19(1): 122, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940073

RESUMO

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.


Assuntos
Proteínas de Bactérias/genética , Citrus/microbiologia , Genoma Bacteriano/genética , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Rhizobiaceae/genética , Animais , Citrus/imunologia , Resistência à Doença , Genótipo , Hemípteros/microbiologia , Insetos Vetores/microbiologia , Floema/imunologia , Floema/microbiologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , RNA Bacteriano/genética , RNA Mensageiro/genética , Rhizobiaceae/fisiologia
5.
Anal Bioanal Chem ; 411(14): 3125-3133, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30989272

RESUMO

Huanglongbing (HLB) or citrus greening is a devastating disease of citrus trees that is caused by the gram-negative Candidatus Liberibacter spp. bacteria. The bacteria are phloem limited and transmitted by the Asian citrus psyllid, Diaphorina citri, and the African citrus psyllid, Trioza erytreae, which allows for a wider dissemination of HLB. Infected trees exhibit yellowing of leaves, premature leaf and fruit drop, and ultimately the death of the entire plant. Polymerase chain reaction (PCR) and antibody-based assays (ELISA and/or immunoblot) are commonly used methods for HLB diagnostics. However, they are costly, time-consuming, and destructive to the sample and often not sensitive enough to detect the pathogen very early in the infection stage. Raman spectroscopy (RS) is a noninvasive, nondestructive, analytical technique which provides insight into the chemical structures of a specimen. In this study, by using a handheld Raman system in combination with chemometric analyses, we can readily distinguish between healthy and HLB (early and late stage)-infected citrus trees, as well as plants suffering from nutrient deficits. The detection rate of Raman-based diagnostics of healthy vs HLB infected vs nutrient deficit is ~ 98% for grapefruit and ~ 87% for orange trees, whereas the accuracy of early- vs late-stage HLB infected is 100% for grapefruits and ~94% for oranges. This analysis is portable and sample agnostic, suggesting that it could be utilized for other crops and conducted autonomously. Graphical abstract.


Assuntos
Citrus/química , Citrus/microbiologia , Nutrientes/análise , Doenças das Plantas/microbiologia , Rhizobiaceae/isolamento & purificação , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Citrus/genética , DNA Bacteriano/análise , DNA de Plantas/análise , Ensaio de Imunoadsorção Enzimática , Nutrientes/deficiência , Folhas de Planta/química , Folhas de Planta/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Rhizobiaceae/genética
6.
PLoS One ; 14(3): e0213535, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870453

RESUMO

Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 µM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Methylococcaceae/crescimento & desenvolvimento , Rhizobiaceae/crescimento & desenvolvimento , Técnicas de Cocultura , Methylococcaceae/genética , Rhizobiaceae/genética
7.
Mol Plant Microbe Interact ; 32(9): 1175-1187, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30925227

RESUMO

Citrus huanglongbing (HLB) is one of the most destructive diseases affecting citrus plants. 'Candidatus Liberibacter asiaticus', an uncultivated α-proteobacteria, is the most widely spread causal agent of HLB and is transmitted by the Asian citrus psyllid Diaphorina citri. 'Ca. L. asiaticus' attachment to the psyllid midgut is believed to be critical to further infect other organs, including the salivary gland. In this study, the type IVc tight adherence (Tad) pilus locus encoded by 'Ca. L. asiaticus' was characterized. The Tad loci are conserved among members of Rhizobiaceae, including 'Ca. L. asiaticus' and Agrobacterium spp. Ectopic expression of the 'Ca. L. asiaticus' cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens, indicating CpaF of 'Ca. L. asiaticus' was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that 'Ca. L. asiaticus' Tad pilus-encoding genes and 'Ca. L. asiaticus' pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that 'Ca. L. asiaticus' VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3. Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the ß-galactosidase reporter gene. Comparative expression analysis confirmed that 'Ca. L. asiaticus' visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing 'Ca. L. asiaticus' visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by 'Ca. L. asiaticus' and shed light on the involvement of VisNR in regulation of the Tad locus.


Assuntos
Proteínas de Bactérias , Citrus , Fímbrias Bacterianas , Regulação Bacteriana da Expressão Gênica , Hemípteros , Rhizobiaceae , Animais , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/genética , Hemípteros/microbiologia , Insetos Vetores/microbiologia , Doenças das Plantas/microbiologia , Rhizobiaceae/genética
8.
PLoS One ; 14(2): e0212020, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794562

RESUMO

Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated "Candidatus Liberibacter spp". "Candidatus Liberibacter spp" that infect Citrus includes "Candidatus Liberibacter asiaticus" (Las), "Candidatus Liberibacter africanus" (Laf) and "Candidatus Liberibacter americanus" (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.


Assuntos
Citrus/microbiologia , Primers do DNA/análise , Rhizobiaceae/isolamento & purificação , China , DNA Bacteriano/genética , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Rhizobiaceae/classificação , Rhizobiaceae/genética , Sensibilidade e Especificidade , Especificidade da Espécie
9.
Plant Dis ; 103(3): 468-474, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30632471

RESUMO

In 2017, potato tubers suspected of being infected with the bacterium 'Candidatus Liberibacter solanacearum' were received from the Animal and Plant Health Inspection Service in the United States. A total of 368 chipping tubers were observed for internal symptoms of zebra chip disease, which is associated with 'Ca. L. solanacearum' infection in the United States, Mexico, Central America, and New Zealand. A single tuber sliced at the stem end showed classic zebra chip symptoms of darkened medullary rays, with streaking and necrotic flecking. The symptomatic tuber was confirmed positive for the bacterium by polymerase chain reaction targeting three different 'Ca. L. solanacearum' genes. Sequence analysis of these three genes, and subsequent BLAST analysis, identified the pathogen with 99, 98, and 97% identity to 'Ca. L. solanacearum' for the 16S ribosomal RNA gene, 50S ribosomal proteins L10/L12 genes, and the outer membrane protein gene, respectively. Sequence analysis did not identify the sample as one of the six known haplotypes of 'Ca. L. solanacearum,' indicating that a seventh haplotype of the pathogen was identified. This new haplotype, designated haplotype F, is now the third haplotype of the bacterium that infects Solanum tuberosum in the United States.


Assuntos
Haplótipos , Rhizobiaceae , Solanum tuberosum , Animais , Genes Bacterianos/genética , Doenças das Plantas/microbiologia , Rhizobiaceae/classificação , Rhizobiaceae/genética , Solanum tuberosum/microbiologia
10.
Methods Mol Biol ; 1864: 37-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30415327

RESUMO

Ensifer adhaerens OV14 underpins the successful crop transformation protocol, termed Ensifer-mediated transformation (EMT). The adaptability and efficiency of EMT technology to successfully transform both monocot and dicots have been previously reported. To facilitate community users' transition to EMT, the modified rice and oilseed rape plants generated in this work were developed using EMT protocols that were grounded in standard Agrobacterium-mediated transformation (AMT) processes. Therefore, this chapter describes simple yet crucial steps involved in transferring the use of EMT of rice and oilseed rape for generation of fertile and independent transgenic lines.


Assuntos
Plantas Geneticamente Modificadas/genética , Rhizobiaceae/genética , Transformação Bacteriana/genética , Animais , Brassica rapa/genética , Vetores Genéticos/genética , Oryza/genética , Sementes , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos
11.
Phytopathology ; 109(4): 551-559, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30303769

RESUMO

Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable bacterium, 'Candidatus Liberibacter asiaticus'. 'Ca. L. asiaticus' in the United States was first found in Florida in 2005 and is now endemic there. In California, 'Ca. L. asiaticus' was first detected in Hacienda Heights in Los Angeles County in 2012 and has now been detected in multiple urban locations in southern California. Knowledge of 'Ca. L. asiaticus' strain diversity in California is important for HLB management. In this study, genomic diversity among 10 'Ca. L. asiaticus' strains from six California locations were analyzed using a next-generation sequencing (NGS) (Illumina MiSeq and HiSeq) approach. Draft genome sequences of 'Ca. L. asiaticus' strains were assembled. Sequences of the 16S ribosomal RNA gene and nrdB confirmed 'Ca. L. asiaticus' identity. Prophages were detected in all 'Ca. L. asiaticus' strains. The California 'Ca. L. asiaticus' strains formed four prophage typing groups (PTGs): PTG1, with type 1 prophage only (strains from Anaheim, San Gabriel, and Riverside); PTG2, with type 2 prophage only (strains from Hacienda Heights); PTG1-3, with both type 1 and 3 prophages (a strain from Cerritos); and PTG1-2, with both type 1 and type 2 prophages (a strain from La Habra). Analyses of the terL sequence showed that all California 'Ca. L. asiaticus' strains were not introduced from Florida but likely from locations in Asia. Miniature inverted-repeat transposable elements were found in all 'Ca. L. asiaticus' strains, yet, a jumping-out event was detected in the 'Ca. L. asiaticus' strain from Cerritos. Altogether, this study demonstrated that the NGS approach focusing on prophage variation was sensitive and effective in revealing diversity of 'Ca. L. asiaticus' strains in California.


Assuntos
Variação Genética , Prófagos , Rhizobiaceae , Ásia , California , Citrus , Florida , Doenças das Plantas , Prófagos/genética , Rhizobiaceae/genética
12.
Microb Ecol ; 78(1): 206-222, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30474731

RESUMO

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of 'Candidatus Liberibacter asiaticus' (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), 'Candidatus Carsonella ruddii,' and 'Candidatus Profftella armatura.' Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem.


Assuntos
Citrus/microbiologia , Endófitos/fisiologia , Hemípteros/microbiologia , Doenças das Plantas/microbiologia , Rhizobiaceae/fisiologia , Simbiose , Animais , Citrus/parasitologia , Endófitos/genética , Endófitos/isolamento & purificação , Feminino , Hemípteros/fisiologia , Insetos Vetores/microbiologia , Insetos Vetores/fisiologia , Masculino , Rhizobiaceae/genética , Rhizobiaceae/isolamento & purificação , Wolbachia/genética , Wolbachia/isolamento & purificação , Wolbachia/fisiologia
13.
J Agric Food Chem ; 67(1): 63-71, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30576131

RESUMO

Biodegradation of pesticide pollution is often restricted by environmental pressures, such as nutrient deprivation. Ensifer adhaerens CGMCC 6315 could overcome this issue and degrade neonicotinoid acetamiprid (ACE) efficiently under low nutrient stimuli. The ACE degradation rate improved by 33.1-fold when the lysogeny broth content for cell culture was decreased to 1/15-fold. Resting cells of CGMCC 6315 degraded 94.4% of 200 mg/L ACE in 12 h and quickly eliminated 87.8% of 5 mg/kg of residual soil ACE within 2 d. ACE degradation by CGMCC 6315 was via a nitrile hydratase (NHase) pathway. Genome sequencing showed that CGMCC 6315 had two NHase genes ( cnhA and pnhA). PnhA had the highest reported activity of 28.8 U/mg for ACE. QPCR and proteomic analysis showed that the improved ACE degradation ability was attributed to the up-regulated expression of PnhA. This biodegradation system of CGMCC 6315 has great potential for use in pesticide pollution remediation.


Assuntos
Inseticidas/metabolismo , Neonicotinoides/metabolismo , Rhizobiaceae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Hidroliases/genética , Hidroliases/metabolismo , Inseticidas/química , Cinética , Neonicotinoides/química , Rhizobiaceae/enzimologia , Rhizobiaceae/genética
14.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30552192

RESUMO

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase ß-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbP Las), mRNA levels of ferR of L. asiaticus (ferR Las) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas The induction of ferR Las expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas We show that the presence of FerR stabilizes and augments the activity of PrbPLas In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.


Assuntos
Ferredoxinas/genética , Ferredoxinas/metabolismo , Rhizobiaceae/genética , Rhizobiaceae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Modelos Moleculares , Pressão Osmótica , Doenças das Plantas/microbiologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
15.
PLoS One ; 13(12): e0208530, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30540789

RESUMO

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismo , Rhizobiaceae/genética , Citrus sinensis/crescimento & desenvolvimento , Citrus sinensis/microbiologia , Primers do DNA/química , Primers do DNA/metabolismo , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Rhizobiaceae/isolamento & purificação
16.
BMC Microbiol ; 18(1): 201, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30497377

RESUMO

BACKGROUND: The aggressive spread of Liberibacter asiaticus, a bacterium closely associated with citrus greening, has given rise to an acute crisis in the citrus industry, making it imperative to expand the scientific knowledge base regarding L. asiaticus. Despite several endeavors to culture L. asiaticus, this bacterium has yet to be maintained in axenic culture, rendering identification and analysis of potential treatment targets challenging. Accordingly, a thorough understanding of biological mechanisms involved in the citrus host-microbe relationship is critical as a means of directing the search for future treatment targets. In this study, we evaluate the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. RESULTS: This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. This work described, for the first time, an enzyme of the L,D-transpeptidase family with moonlighting enzyme activity directed to the modification of the bacterial cell wall and LPS. CONCLUSIONS: Taken together, the data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Peptidil Transferases/isolamento & purificação , Peptidil Transferases/metabolismo , Rhizobiaceae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Parede Celular/enzimologia , Parede Celular/genética , Lipopolissacarídeos/metabolismo , Peptidoglicano/metabolismo , Peptidil Transferases/química , Peptidil Transferases/genética , Periplasma/enzimologia , Periplasma/genética , Periplasma/metabolismo , Transporte Proteico , Rhizobiaceae/química , Rhizobiaceae/genética
17.
PLoS One ; 13(10): e0204702, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30304000

RESUMO

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide.


Assuntos
Albuminas 2S de Plantas/administração & dosagem , Antibacterianos/administração & dosagem , Citrus/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Rhizobiaceae/efeitos dos fármacos , Óxido de Zinco/administração & dosagem , Animais , Carga Bacteriana/efeitos dos fármacos , Carga Bacteriana/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Hemípteros/microbiologia , Insetos Vetores/microbiologia , Nanoestruturas/administração & dosagem , Pós , Rhizobiaceae/genética , Rhizobiaceae/crescimento & desenvolvimento
18.
J Mol Microbiol Biotechnol ; 28(3): 116-127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30176677

RESUMO

Citrus greening disease, or huanglongbing, may entirely eradicate all varieties of citrus cultivars worldwide in the near future. This disease is caused by non-cultivable bacteria of the genus Liberibacter; among them, the more pathogenic being Liberibacter asiaticus. The complexity of the host-pathogen relationship, associated with the impossibility of performing research using axenic cultures, has severely hindered the basic research on microbiology. Since its genome sequence was published in 2009, most of the scientific publications in the field were dedicated to in silico analysis and selection of targets to design early detection methods. The knowledge gained with these approaches felt short to articulate effective methods to control the disease progression. There is a critical need to understand the basic biology of bacteria to design effective strategies to inactivate central mechanisms of pathogenesis. In this review, we summarize the scientific progress made by studying L. asiaticus' biology through direct experimentation. The evidence collected thus far is not enough to understand L. -asiaticus' fundamental biology. It is imperiously necessary to increase the basic research to identify relevant biological clues to control citrus greening. The gained knowledge may also help to prevent potential catastrophic diseases in other crops of significant importance caused by other unculturable Liberibacter species.


Assuntos
Doenças das Plantas/microbiologia , Rhizobiaceae/genética , Rhizobiaceae/fisiologia , Rhizobiaceae/patogenicidade , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sequência de Bases , Proteínas de Transporte/genética , Citrus , Genoma Bacteriano , Genômica , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Membrana , Metagenômica , Translocases Mitocondriais de ADP e ATP , Peroxidase , Peroxirredoxinas , Imunidade Vegetal , Prófagos , Proteômica , Serina Endopeptidases , Transcriptoma , Sistemas de Secreção Tipo V , Zinco/metabolismo
19.
FEMS Microbiol Ecol ; 94(11)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30184201

RESUMO

Nodules of Chamaecrista pumila growing in several locations in India were sampled for anatomical studies and for characterization of their rhizobial microsymbionts. Regardless of their region of origin, the nodules were indeterminate with their bacteroids contained within symbiosomes which were surrounded by pectin. More than 150 strains were isolated from alkaline soils from the Thar Desert (Rajasthan), wet-acidic soils of Shillong (Meghalaya), and from trap experiments using soils from four other states with different agro-ecological regions. Molecular phylogenetic analysis based on five housekeeping (rrs, recA, glnII, dnaK andatpD) and two symbiotic (nodA and nifH) genes was performed for selected strains. Chamaecrista pumila was shown to be nodulated by niche-specific diverse strains of either Ensifer or Bradyrhizobium in alkaline (Thar Desert) to neutral (Tamil Nadu) soils and only Bradyrhizobium strains in acidic (Shillong) soils. Concatenated core gene phylogenies showed four novel Ensifer-MLSA types and nine Bradyrhizobium-MLSA types. Genetically diverse Ensifer strains harbored similar sym genes which were novel. In contrast, significant symbiotic diversity was observed in the Bradyrhizobium strains. The C. pumila strains cross-nodulated Vigna radiata and some wild papilionoid and mimosoid legumes. It is suggested that soil pH and moisture level played important roles in structuring the C. pumila microsymbiont community.


Assuntos
Bradyrhizobium/isolamento & purificação , Chamaecrista/microbiologia , Rhizobiaceae/isolamento & purificação , Microbiologia do Solo , Bradyrhizobium/classificação , Bradyrhizobium/genética , Chamaecrista/anatomia & histologia , Chamaecrista/ultraestrutura , Clima , Concentração de Íons de Hidrogênio , Índia , Filogenia , Rhizobiaceae/classificação , Rhizobiaceae/genética , Nódulos Radiculares de Plantas/anatomia & histologia , Nódulos Radiculares de Plantas/ultraestrutura , Solo/química , Simbiose/genética
20.
Syst Appl Microbiol ; 41(6): 641-649, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30145046

RESUMO

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.


Assuntos
Bradyrhizobiaceae/classificação , Fabaceae/microbiologia , Mineração , Rhizobiaceae/classificação , Nódulos Radiculares de Plantas/microbiologia , Agricultura , Austrália , Bradyrhizobiaceae/genética , Bradyrhizobiaceae/isolamento & purificação , DNA Bacteriano/genética , Genes Bacterianos , Fosfatos , Filogenia , RNA Ribossômico 16S/genética , Rhizobiaceae/genética , Rhizobiaceae/isolamento & purificação , Simbiose
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA