RESUMO
Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 µM added iron (Fe+2 ). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.
Assuntos
Antibiose/genética , Antifúngicos/metabolismo , Ascomicetos/crescimento & desenvolvimento , Pseudomonas/metabolismo , Tropolona/análogos & derivados , Antibiose/fisiologia , Argentina , Proteínas de Bactérias/genética , Mutagênese/efeitos dos fármacos , Pseudomonas/genética , Fatores de Transcrição/genética , Transposases/genética , Tropolona/metabolismoRESUMO
Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.
Assuntos
Hypocrea/enzimologia , Hypocrea/genética , Proteínas ras/genética , Proteínas ras/metabolismo , Antibiose/genética , Basidiomycota/crescimento & desenvolvimento , Agentes de Controle Biológico , DNA Fúngico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Hypocrea/crescimento & desenvolvimento , Interações Microbianas/genética , Mutação , Filogenia , Doenças das Plantas/microbiologia , Rhizoctonia/crescimento & desenvolvimento , Metabolismo Secundário/genética , Esporos Fúngicos/genéticaRESUMO
Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.
Assuntos
Antibiose/genética , Parede Celular/enzimologia , Parede Celular/metabolismo , Endo-1,3(4)-beta-Glucanase/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Ascomicetos/metabolismo , Benomilo/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Quitina/metabolismo , Endo-1,3(4)-beta-Glucanase/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genômica , Microscopia de Fluorescência , Filogenia , Rhizoctonia/metabolismo , Trichoderma/efeitos dos fármacos , Trichoderma/patogenicidade , beta-Glucanas/metabolismoRESUMO
Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.
Assuntos
Proteínas de Bactérias/fisiologia , Stenotrophomonas maltophilia/fisiologia , Stenotrophomonas maltophilia/patogenicidade , Sistemas de Secreção Tipo IV/fisiologia , Sequência de Aminoácidos , Antibiose/genética , Antibiose/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Genes Bacterianos , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Proteínas Reguladoras de Ferro/química , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/fisiologia , Modelos Moleculares , Infecções Oportunistas/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Stenotrophomonas maltophilia/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimentoRESUMO
Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5'-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Genoma Viral , Fagos de Streptococcus/genética , Fagos de Streptococcus/patogenicidade , Streptococcus thermophilus/genética , Antibiose/genética , Sequência de Bases , Queijo/microbiologia , Queijo/virologia , Mapeamento Cromossômico , DNA Intergênico/genética , Fermentação , Tecnologia de Alimentos/economia , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anotação de Sequência Molecular , Mutação , Alinhamento de Sequência , Fagos de Streptococcus/ultraestrutura , Streptococcus thermophilus/imunologia , Streptococcus thermophilus/virologia , Uruguai , VirulênciaRESUMO
Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (p<0.05) up-regulation of ALS3, HWP1, SAP2 and SAP6, and hyphal production occurred in biofilms containing streptococci (Ca-Ss-Sm). In Ca-Pg biofilms, down-regulation of HWP1 and SAP4 expression, with reduced hyphal production occurred. Ca-Ss-Sm-Pg biofilms had increased hyphal proportions and up-regulation of ALS3, SAP2 and SAP6. In conclusion, C. albicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans , Hifas/crescimento & desenvolvimento , Porphyromonas gingivalis/fisiologia , Streptococcus sanguis/fisiologia , Titânio , Antibiose/genética , Candida albicans/patogenicidade , Candida albicans/fisiologia , Proteínas Fúngicas/genética , Humanos , Glicoproteínas de Membrana/genética , Peri-Implantite/microbiologia , Propriedades de Superfície , Virulência , Fatores de Virulência/metabolismoRESUMO
O objetivo deste trabalho foi identificar cultivares de caupi resistentes a B. tabaci biótipo B e os respectivos tipos de resistência envolvidos. Os experimentos foram conduzidos em casa de vegetação no Departamento de Fitossanidade da Faculdade de Ciências Agrárias e Veterinárias, UNESP/Campus de Jaboticabal, SP, no período de novembro de 2009 a abril de 2010. Realizaramse testes de não preferência para oviposição e antibiose. O delineamento experimental utilizado foi de blocos casualizados para o teste com chance de escolha e inteiramente casualizado para os testes sem chance de escolha e teste de antibiose. Conclui-se que os cultivares BRS Urubuquara, IPA-206 e BR17 Gurgueia apresentaram resistência do tipo não preferência para oviposição da mosca-branca; a cultivar BRS Urubuquara apresenta resistência de tipo antibiose; a cultivar Sempre Verde é suscetível; e, mosca-branca prefere ovipositar na posição superior das plantas. (AU)
The objective of this study was to identify cowpea cultivars resistant to B. tabaci biotype B and the respective resistance types involved. The experiments were conducted in a greenhouse at the Departamento de Fitossanidade da Faculdade de Ciências Agrárias e Veterinárias, UNESP/Campus de Jaboticabal, SP, Brasil, from November 2009 to April 2010. Tests for non-preference for oviposition and antibiosis were performed. The experimental design consisted of randomized blocks for the choice test and completely randomized for the no-choice tests and the antibiosis test. It was concluded that the BRS Urubuquara, IPA-206 and BR17 Gurgueia cultivars present non-preference for whitefly oviposition resistance; the BRS Urubuquara cultivar presents nonpreference and antibiosis resistance; the Sempre Verde cultivate is susceptible to oviposition; and the whitefly prefers to deposit eggs on the leaves abaxial face in the upper position of the plant. (AU)
Assuntos
Animais , Antibiose/genética , Oviposição , Fabaceae , Dípteros/classificação , Comportamento AnimalRESUMO
O objetivo deste trabalho foi identificar cultivares de caupi resistentes a B. tabaci biótipo B e os respectivos tipos de resistência envolvidos. Os experimentos foram conduzidos em casa de vegetação no Departamento de Fitossanidade da Faculdade de Ciências Agrárias e Veterinárias, UNESP/Campus de Jaboticabal, SP, no período de novembro de 2009 a abril de 2010. Realizaramse testes de não preferência para oviposição e antibiose. O delineamento experimental utilizado foi de blocos casualizados para o teste com chance de escolha e inteiramente casualizado para os testes sem chance de escolha e teste de antibiose. Conclui-se que os cultivares BRS Urubuquara, IPA-206 e BR17 Gurgueia apresentaram resistência do tipo não preferência para oviposição da mosca-branca; a cultivar BRS Urubuquara apresenta resistência de tipo antibiose; a cultivar Sempre Verde é suscetível; e, mosca-branca prefere ovipositar na posição superior das plantas.
The objective of this study was to identify cowpea cultivars resistant to B. tabaci biotype B and the respective resistance types involved. The experiments were conducted in a greenhouse at the Departamento de Fitossanidade da Faculdade de Ciências Agrárias e Veterinárias, UNESP/Campus de Jaboticabal, SP, Brasil, from November 2009 to April 2010. Tests for non-preference for oviposition and antibiosis were performed. The experimental design consisted of randomized blocks for the choice test and completely randomized for the no-choice tests and the antibiosis test. It was concluded that the BRS Urubuquara, IPA-206 and BR17 Gurgueia cultivars present non-preference for whitefly oviposition resistance; the BRS Urubuquara cultivar presents nonpreference and antibiosis resistance; the Sempre Verde cultivate is susceptible to oviposition; and the whitefly prefers to deposit eggs on the leaves abaxial face in the upper position of the plant.
Assuntos
Animais , Antibiose/genética , Fabaceae , Oviposição , Comportamento Animal , Dípteros/classificaçãoRESUMO
The pathogenic fungus Fusarium graminearum is an ongoing threat to agriculture, causing losses in grain yield and quality in diverse crops. Substantial progress has been made in the identification of genes involved in the suppression of phytopathogens by antagonistic microorganisms; however, limited information regarding responses of plant pathogens to these biocontrol agents is available. Gene expression analysis was used to identify differentially expressed transcripts of the fungal plant pathogen F. graminearum under antagonistic effect of the bacterium Pantoea agglomerans. A macroarray was constructed, using 1014 transcripts from an F. graminearum cDNA library. Probes consisted of the cDNA of F. graminearum grown in the presence and in the absence of P. agglomerans. Twenty-nine genes were either up (19) or down (10) regulated during interaction with the antagonist bacterium. Genes encoding proteins associated with fungal defense and/or virulence or with nutritional and oxidative stress responses were induced. The repressed genes coded for a zinc finger protein associated with cell division, proteins containing cellular signaling domains, respiratory chain proteins, and chaperone-type proteins. These data give molecular and biochemical evidence of response of F. graminearum to an antagonist and could help develop effective biocontrol procedures for pathogenic plant fungi.
Assuntos
Antibiose/genética , Produtos Agrícolas/microbiologia , Fusarium/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Pantoea/fisiologia , Regulação para Baixo/genética , Fusarium/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genéticaRESUMO
Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested soil significantly reduced the disease caused by R. solani in cotton plants.