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1.
Curr Microbiol ; 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32185467

RESUMO

Many studies have focused on the significant role of biofilm formation by Bacillus in the biocontrol process. Bacillus pumilus HR10 is a plant growth-promoting rhizobacterium with multiple biocontrol functions, including promoting growth, controlling pathogens, and assisting in the formation of mycorrhizae. Currently, there is no relevant report on the biofilm formation of B. pumilus HR10 and its influencing factors. B. pumilus HR10 was found to easily form a stable biofilm structure on the surface of media, with awesome swarming ability. The optimum temperature for biofilm formation was 37 °C. B. pumilus HR10 formed most obviously at pH 7.0 and was not extremely sensitive to acidic or alkaline conditions. Most of the polysaccharide components of plant root exudates promoted the biofilm formation by B. pumilus HR10, with glucose having the greatest promoting effect. Low concentrations of Fe2+, Mg2+, Ca2+, K+, and Na+ enhanced biofilm formation. In summary, biofilm formation can improve the tolerance of B. pumilus HR10 to salt and certain heavy metal ion stresses and contribute to its application in different plants and soils with high salinity or heavy metals in the field.

2.
Front Microbiol ; 10: 2559, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31749788

RESUMO

Burkholderia pyrrocinia JK-SH007 is a plant growth-promoting bacteria (PGPB), that can promote the growth of poplar and other trees, and, production of the plant hormone indole-3-acetic acid (IAA) is one of the reasons for this effect. Therefore, the aims of this study were to evaluate the effect of the external environment on the synthesis of IAA by B. pyrrocinia JK-SH007 and to perform a functional analysis of its IAA synthesis pathway. In this study, IAA and its synthetic intermediates indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), tryptamine (TAM), and indole-3-acetonitrile (IAN) were detected in B. pyrrocinia JK-SH007 fermentation broth by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS), and these indolic compounds were also found in the cell-free extraction of B. pyrrocinia JK-SH007, but the genomic analysis of B. pyrrocinia JK-SH007 indicated that IAA biosynthesis was mainly through the IAM and TAM pathways. The effects of L-tryptophan (L-Trp), temperature and pH on the synthesis of IAA were investigated, and the results showed that L-Trp exerted a significant effect on IAA synthesis and that 37°C and pH 7 were the optimal conditions IAA production by B. pyrrocinia JK-SH007. In addition, the protein expression of tryptophan 2-monooxygenase and indoleacetamide hydrolase, which are the key enzymes of the indole acetamide-mediated IAA synthesis pathway, was analyzed, and their activity was verified by substrate feeding experiments. The results revealed the existence of an IAA synthesis pathway mediated by IAM and indicated that this pathway plays a role in B. pyrrocinia JK-SH007. This study lays the foundation for further exploration of the specific pathway and mechanism of IAA synthesis in B. pyrrocinia JK-SH007.

4.
Exp Parasitol ; 197: 51-56, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30641035

RESUMO

The pine wood nematode Bursaphelenchus xylophilus is a disastrous pathogen of pine forests in East Asia and Europe. Despite its decimating effect on pine forests, efficient and environmentally friendly methods available to control the pine wood nematode (PWN) are limited. The most abundant protein in nematode sperm, major sperm proteins (MSPs) have only been discovered in nematodes. In this study, phylogenetic analysis showed that BxMSP10 was highly conserved in the nematode and had a closer phylogenetic relationship with free-living nematodes than with plant-parasitic nematode species. BxMSP10 was specifically expressed in the seminal vesicle of male adults. dsRNA of BxMSP10 significantly decreased reproduction, egg hatching and population maintenance in B. xylophilus. These results indicated that BxMSP10 was a potential candidate for application in the control of B. xylophilus.


Assuntos
Proteínas de Helminto/fisiologia , Rabditídios/fisiologia , Animais , Botrytis , DNA de Helmintos/isolamento & purificação , Feminino , Expressão Gênica , Hordeum/microbiologia , Hordeum/parasitologia , Hibridização In Situ , Íntrons , Masculino , Filogenia , Pinus/parasitologia , Doenças das Plantas/parasitologia , Interferência de RNA , RNA de Helmintos/isolamento & purificação , RNA de Helmintos/fisiologia , Reprodução/fisiologia , Rabditídios/classificação , Rabditídios/genética
5.
Mol Plant Microbe Interact ; 32(4): 452-463, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30351223

RESUMO

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.


Assuntos
Pinus , Tylenchida , Virulência , Animais , Morte Celular , China , Pinus/parasitologia , Tylenchida/genética , Tylenchida/patogenicidade , Virulência/genética
6.
Genes (Basel) ; 9(11)2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30413120

RESUMO

Mitogen-activated protein kinase (MAPK) signaling pathway plays key roles in sensing extracellular signals and transmitting them from the cell membrane to the nucleus in response to various environmental stimuli. A MAPKKK protein CgMck1 in Colletotrichum gloeosporioides was characterized. Phenotypic analyses of the ∆Cgmck1 mutant showed that the CgMck1 was required for vegetative growth, fruiting body development, and sporulation. Additionally, the CgMCK1 deletion mutant showed significant defects in cell wall integrity, and responses to osmotic stresses. The mutant abolished the ability to develop appressorium, and lost pathogenicity to host plants. The ∆Cgmck1 mutant also exhibited a higher sensitivity to antifungal bacterium agent Bacillus velezensis. The deletion mutants of downstream MAPK cascades components CgMkk1 and CgMps1 showed similar defects to the ∆Cgmck1 mutant. In conclusion, CgMck1 is involved in the regulation of vegetative growth, asexual development, cell wall integrity, stresses resistance, and infection morphogenesis in C. gloeosporioides.

7.
Front Microbiol ; 9: 2311, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405539

RESUMO

Potassium has an important role to play in multiple cellular processes. In Saccharomyces cerevisiae, the serine/threonine (S/T) kinase Sat4/Hal4 is required for potassium accumulation, and thus, regulates the resistance to sodium salts and helps in the stabilization of other plasma membrane transporters. However, the functions of Sat4 in filamentous phytopathogenic fungi are largely unknown. In this study, ChSat4, the yeast Sat4p homolog in Colletotrichum higginsianum, has been identified. Target deletion of ChSAT4 resulted in defects in mycelial growth and sporulation. Intracellular K+ accumulation was significantly decreased in the ChSAT4 deletion mutant. Additionally, the ΔChsat4 mutant showed defects in cell wall integrity, hyperoxide stress response, and pathogenicity. Localization pattern analysis indicated ChSat4 was localized in the cytoplasm. Furthermore, ChSat4 showed high functional conservation with the homolog FgSat4 in Fusarium graminearum. Taken together, our data indicated that ChSat4 was important for intracellular K+ accumulation and infection morphogenesis in C. higginsianum.

8.
PLoS One ; 13(7): e0199625, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29995910

RESUMO

Phosphate-solubilizing fungi (PSF) generally enhance available phosphorus (P) released from soil, which contributes to plants' P requirement, especially in P-limiting regions. In this study, two PSF, TalA-JX04 and AspN-JX16, were isolated from the rhizosphere soil of moso bamboo (Phyllostachys edulis) widely distributed in P-deficient areas in China and identified as Talaromyces aurantiacus and Aspergillus neoniger, respectively. The two PSF were cultured in potato dextrose liquid medium with six types of initial pH values ranging from 6.5 to 1.5 to assess acid resistance. Both PSF were incubated in Pikovskaya's liquid media with different pH values containing five recalcitrant P sources, including Ca3(PO4)2, FePO4, CaHPO4, AlPO4, and C6H6Ca6O24P6, to estimate their P-solubilizing capacity. No significant differences were found in the biomass of both fungi grown in media with different initial pH, indicating that these fungi could grow well under acid stress. The P-solubilizing capacity of TalA-JX04 was highest in medium containing CaHPO4, followed by Ca3(PO4)2, FePO4, C6H6Ca6O24P6, and AlPO4 in six types of initial pH treatments, while the recalcitrant P-solubilizing capacity of AspN-JX16 varied with initial pH. Meanwhile, the P-solubilizing capacity of AspN-JX16 was much higher than TalA-JX04. The pH of fermentation broth was negatively correlated with P-solubilizing capacity (p<0.01), suggesting that the fungi promote the dissolution of P sources by secreting organic acids. Our results showed that TalA-JX04 and AspN-JX16 could survive in acidic environments and both fungi had a considerable ability to release soluble P by decomposing recalcitrant P-bearing compounds. The two fungi had potential for application as environment-friendly biofertilizers in subtropical bamboo ecosystem.


Assuntos
Fungos/metabolismo , Concentração de Íons de Hidrogênio , Fosfatos/metabolismo , Fósforo/metabolismo , Rizosfera , Sasa/microbiologia , Microbiologia do Solo , Análise de Variância , Biomassa , Fungos/isolamento & purificação , Micélio , Fenótipo
9.
Biomed Res Int ; 2018: 9619724, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30687759

RESUMO

The objective of this study was to investigate whether plant-bacteria interaction affects the secretion of organic acids by both organisms and to assess whether the production of IAA by the bacterium increases the secretion of organic acids by root exudates, and if the stress produced by low available phosphorus (P) affects the production of organic acids by bacteria, by roots, or by root exudates in presence of bacterial cultures. With this purpose, we used as a biological model poplar plants and one strain of Burkholderia multivorans able to solubilize P. High performance liquid chromatography was utilized to measure organic acids. The tests, the inductive effects of exogenous indole-3-acetic acid (IAA) on secretion of organic acids, the 2 × 4 × 2 factorial design experiment, and the ability of organic acids to solubilize tricalcium phosphate were performed to investigate the interactive effects. The results showed that, after B. multivorans WS-FJ9 interacted with the poplar root system, the key phosphate-solubilizing driving force was gluconic acid (GA) which was produced in three ways: (1) secreted by the root system in the presence of IAA produced by B. multivorans WS-FJ9; (2) secreted by B. multivorans WS-FJ9; and (3) secreted by the poplar root system in the presence of phosphorus stress. When phosphorus stress was absent, the GA was produced as outlined in (1) and (2) above. These results demonstrated that inoculating B. multivorans WS-FJ9 into the poplar root system could increase the amount of GA secretion and implied that the interaction between B. multivorans WS-FJ9 and the poplar root system could contribute to the increase of P available fraction for poplar plants.


Assuntos
Complexo Burkholderia cepacia/metabolismo , Compostos Orgânicos/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plantas/microbiologia , Fosfatos/metabolismo , Fósforo/metabolismo
10.
Plant Dis ; 102(3): 500-506, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30673483

RESUMO

Chinese fir (Cunninghamia lanceolata) is a significant timber species that has been broadly cultivated in southern China. A shoot blight disease on Chinese fir seedlings was discovered in Fujian, China and a fungus was then consistently associated with the symptoms. This fungus was determined to be causing this disease, among others by fulfilling Koch's postulates. Based on morphological characteristics and multilocus phylogenetic analyses with the sequences of the internal transcribed spacer, partial glyceraldehyde-3-phosphate dehydrogenase gene, partial translation elongation factor 1-α gene, and partial 28S large subunit ribosomal RNA gene, the fungus was identified as Bipolaris oryzae. These characteristics and phylogenetic analyses clearly support that this pathogen is different from B. sacchari, which was, until now, considered to be the causal agent of a similar blight on Chinese fir in Guangdong, China. The fungus was also shown to be strongly pathogenic to rice, one of the most susceptible hosts to B. oryzae. Crop rotation involving rice is often carried out with Chinese fir in southern China, a practice that most likely increases the risk of shoot blight on C. lanceolata. To our knowledge, shoot blight caused by B. oryzae is reported for the first time in a gymnosperm species.


Assuntos
Ascomicetos/isolamento & purificação , Cunninghamia/microbiologia , Doenças das Plantas/microbiologia , Ascomicetos/citologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Tipagem de Sequências Multilocus , Técnicas de Tipagem Micológica , Filogenia , Brotos de Planta/microbiologia , Plântula/microbiologia
11.
Int J Mol Sci ; 18(12)2017 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-29211016

RESUMO

The pine wood nematode (PWN), Bursaphelenchusxylophilus, is the pathogen of pine wilt disease (PWD) and causes huge economic losses in pine forests and shows a remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B.xylophilus. Our previous studies showed that autophagy exists in PWN. Therefore, we tested the effects of autophagy inducer rapamycin on PWN and the results revealed that the feeding rate and reproduction were significantly promoted on fungal mats. The gene expression patterns of BxATG1 and BxATG8 under the different stress were determined by quantitative reverse transcription PCR (qRT-PCR). We tested the effects of RNA interference on BxATG1 and BxATG8 in PWN during different periods of infection in Pinus thunbergii. The results revealed that BxATG1 and BxATG8 may play roles in allowing PWN to adapt to changing environmental conditions and the virulence of PWN was influenced by the silence of autophagy-related genes BxATG1 and BxATG8. These results provided fundamental information on the relationship between autophagy and PWN, and on better understanding of gene function of BxATG1 and BxATG8 in PWN.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Helminto/metabolismo , Nematoides/metabolismo , Estresse Fisiológico , Adaptação Fisiológica , Animais , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas de Helminto/genética , Nematoides/genética , Nematoides/patogenicidade , Pinus/parasitologia , Virulência
12.
J Microbiol Methods ; 132: 95-98, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27725176

RESUMO

Burkholderia pyrrocinia strain JK-SH007 isolated from poplar stems plays a highly significant role in the growth promotion and the biocontrol of poplar canker during colonization in poplar. In this research, the ideal reference gene was filtered and determined for the transcript normalization. Additionally, the expression of pyrG under all four conditions was relatively stable in B. pyrrocinia JK-SH007.


Assuntos
Proteínas de Bactérias/genética , Burkholderia/genética , Burkholderia/isolamento & purificação , Genes Bacterianos , Reação em Cadeia da Polimerase em Tempo Real , Burkholderia/classificação
13.
Int J Mol Sci ; 17(3): 279, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26950119

RESUMO

The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/citologia , Tylenchida/genética , Animais , Interações Hospedeiro-Parasita , Interferência de RNA , Tylenchida/fisiologia
14.
Int J Mol Sci ; 17(1)2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26797602

RESUMO

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD.


Assuntos
Inativação Gênica , Proteínas de Helminto/antagonistas & inibidores , Pinus/parasitologia , Polissacarídeo-Liase/antagonistas & inibidores , Tylenchida/genética , Fatores de Virulência/antagonistas & inibidores , Animais , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Doenças das Plantas/parasitologia , Doenças das Plantas/terapia , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Tylenchida/enzimologia , Tylenchida/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Madeira/parasitologia
15.
Plant Dis ; 100(4): 672-678, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30688606

RESUMO

The genus Colletotrichum is considered the eighth most important group of plant-pathogenic fungi in the world due to its scientific and economic importance. Colletotrichum spp. cause anthracnose disease in a wide range of economically important plants. Euonymus japonicus Thunb. (Celastraceae) is a broad-leaved evergreen tree that is widely planted in the parks and landscapes of China. An anthracnose occurs on E. japonicus in China but there has been a disagreement on the identity of the fungal pathogen. In this study, the fungal isolate HYCG2-3 was determined by Koch's postulates to be a pathogen on E. japonicus. Based on the morphological and molecular methods, isolate HYCG2-3 was identified as Colletotrichum gloeosporioides sensu stricto within the C. gloeosporioides species complex.

16.
Int J Mol Sci ; 16(3): 5216-34, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25756378

RESUMO

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Doenças das Plantas/parasitologia , Tylenchida/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Enzimático do Citocromo P-450/metabolismo , Dados de Sequência Molecular , Praguicidas/toxicidade , Pinus/crescimento & desenvolvimento , Pinus/parasitologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Reprodução/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Plântula/parasitologia , Alinhamento de Sequência , Análise de Sequência de DNA , Tylenchida/classificação , Tylenchida/metabolismo , Tylenchida/fisiologia
17.
Gene ; 555(2): 346-56, 2015 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-25447893

RESUMO

Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs and novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Functional analysis revealed that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes. In addition, another set of atypical miRNAs, termed mirtrons, was also identified in this study. Thus, our research has provided detailed characterization of B. xylophilus miRNA expression patterns during the pathological process of pine wilt disease. These findings would contribute to more in-depth understanding of this devastating plant disease.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/metabolismo , Nematoides/genética , Pinus/parasitologia , Doenças das Plantas/parasitologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Biologia Computacional , Sequência Conservada , Evolução Molecular , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/metabolismo , Homologia de Sequência de Aminoácidos
18.
Int J Mol Sci ; 15(6): 10215-32, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24918285

RESUMO

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode.


Assuntos
Peroxirredoxinas/metabolismo , Tylenchida/metabolismo , Animais , Eletroforese em Gel Bidimensional , Escherichia coli/metabolismo , Hibridização In Situ , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Pinus/crescimento & desenvolvimento , Pinus/parasitologia , Doenças das Plantas/parasitologia , Proteoma/análise , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Appl Microbiol Biotechnol ; 97(24): 10489-98, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24092012

RESUMO

Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting biological characteristics, which make them simultaneously beneficial and harmful to humans. They have been exploited for biocontrol, bioremediation, and plant growth promotion. However, their capacity as opportunistic bacteria that infect humans restricts their biotechnological applications. Therefore, the risks of using these bacteria should be assessed. In this study, Burkholderia multivorans WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient in solubilizing phosphate, was evaluated with respect to its biosafety, colonization in poplar rhizosphere, and growth-promoting effects on poplar seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined experimentally using onion and tobacco as model plants. Onion bulb inoculated with B. multivorans WS-FJ9 showed slight hypersensitive responses around the inoculation points, but effects were not detectable based on the inner color and odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited slightly water-soaked spots around the inoculation points, which did not expand or develop into lesions even with repeated incubation. Pathogenicity of the strain in alfalfa, which has been suggested as an alternative Bcc model for mice, was not detectable. Results from gene-specific polymerase chain reactions showed that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA virulence genes. Scanning electron microscopy revealed that the colonization of the WS-FJ9 strain reached 1.4 × 10(4) colony forming units (cfu) g(-1) rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain could colonize the rhizosphere as well as the root tissues and cells of poplars. Greenhouse evaluations in both sterilized and non-sterilized soils indicated that B. multivorans WS-FJ9 significantly promoted growth in height, root collar diameter, and plant biomass of inoculated poplar seedlings compared with controls. Phosphorus contents of roots and stems of treated seedlings were 0.57 and 0.55 mg g(-1) higher than those of the controls, respectively. Phosphorus content was lower in the rhizosphere soils by an average of 1.03 mg g(-1) compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a nonpathogenic strain that could colonize the roots and significantly promote the growth of poplar seedlings.


Assuntos
Complexo Burkholderia cepacia/crescimento & desenvolvimento , Complexo Burkholderia cepacia/patogenicidade , Populus/crescimento & desenvolvimento , Populus/microbiologia , Animais , Carga Bacteriana , Biomassa , Complexo Burkholderia cepacia/genética , Contagem de Colônia Microbiana , Humanos , Medicago sativa/microbiologia , Camundongos , Microscopia Eletrônica de Varredura , Cebolas/microbiologia , Fósforo/análise , Doenças das Plantas/microbiologia , Raízes de Plantas/química , Raízes de Plantas/microbiologia , Caules de Planta/química , Microbiologia do Solo , Tabaco/microbiologia , Fatores de Virulência/genética
20.
Ying Yong Sheng Tai Xue Bao ; 24(3): 646-52, 2013 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-23755476

RESUMO

In the interaction between Pinus thunbergii and Bursaphelenchus xylophilus, nitric oxide (NO) is an important signaling molecule involving in the early response of P. thunbergii to the invasion of B. xylophilus. However, it is unclear that whether the NO production by P. thunbergii is triggered by the invaded B. xylophilus or its secreted metabolites. In the present study, the P. thunbergii was inoculated with living B. xylophilus, its secretion, and the suspension of grinded B. xylophilus, respectively, and the nitric oxide synthase (NOS) activity and NO content in the P. thunbergii were detected at the early stage. In all treatments, the inoculated P. thunbergii appeared disease symptoms, and the NOS activity and NO content in the P. thunbergii inoculated with B. xylophilus secretion and grinded B. xylophilus suspension increased, suggesting that besides living B. xylophilus, its contents or secretion could also trigger the expression of NO response signal in P. thunbergii, inducing the downstream response and causing the disease development of P. thunbergi. With the increasing temperature at 15-25 degrees C, both the NOS activity and the NO content in inoculated P. thunbergii increased, and the disease symptoms appeared earlier. The same patterns of NOS activity, NO content, and disease symptoms were also observed under increasing drought stress. It was suggested that within a definite range, increased. temperature and drought stress could enhance the NO signal expression in inoculated P. thunbergii and accelerate its disease development, and thus, the disease development of inoculated P. thunbergii under high temperature and drought condition could be related to the enhancement of the NO response signal in the host.


Assuntos
Nematoides/patogenicidade , Óxido Nítrico/metabolismo , Pinus/metabolismo , Pinus/parasitologia , Doenças das Plantas/parasitologia , Animais , Óxido Nítrico Sintase/metabolismo
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