RESUMO
Understanding how environmental variables influence biofilm formation becomes relevant for managing Vibrio biofilm-related infections in shrimp production. Therefore, we evaluated the impact of temperature, time, and initial inoculum in the biofilm development of these two Vibrio species using a multifactorial experimental design. Planktonic growth inhibition and inhibition/eradication of Vibrio biofilms, more exactly V. parahaemolyticus (VP87 and VP275) and V. cholerae (VC112) isolated from shrimp farms were evaluated by Eucalyptus and Guava aqueous leaf extracts and compared to tetracycline and ceftriaxone. Preliminary results showed that the best growth conditions of biofilm development for V. parahaemolyticus were 24 h and 24°C (p <0.001), while V. cholerae biofilms were 72 h and 30°C (p <0.001). Multivariate linear regression ANOVA was applied using colony-forming unit (CFU) counting assays as a reference, and R-squared values were applied as goodness-of-fit measurements for biofilm analysis. Then, both plant extracts were analyzed with HPLC using double online detection by diode array detector (DAD) and mass spectrometry (MS) for the evaluation of their chemical composition, where the main identified compounds for Eucalyptus extract were cypellogin A, cypellogin B, and cypellocarpin C, while guavinoside A, B, and C compounds were the main compounds for Guava extract. For planktonic growth inhibition, Eucalyptus extract showed its maximum effect at 200 µg/mL with an inhibition of 75% (p < 0.0001) against all Vibrio strains, while Guava extract exhibited its maximum inhibition at 1600 µg/mL with an inhibition of 70% (p < 0.0001). Both biofilm inhibition and eradication assays were performed by the two conditions (24 h at 24°C and 72 h at 30°C) on Vibrio strains according to desirability analysis. Regarding 24 h at 24°C, differences were observed in the CFU counting between antibiotics and plant extracts, where both plant extracts demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values (Eucalyptus extract: 1600, 3200, and 6400 µg/mL; while Guava extract: 12800, 25600, and 52000 µg/mL). Concerning 72 h at 30°C, results showed a less notorious biomass inhibition by Guava leaf extract and tetracycline. However, Eucalyptus extract significantly reduced the total number of viable cells within Vibrio biofilms from 2x to 32x MIC values (400-6400 µg/mL) when compared to the same MIC values of ceftriaxone (5-80 µg/mL), which was not able to reduce viable cells. Eucalyptus extract demonstrated similar results at both growth conditions, showing an average inhibition of approximately 80% at 400 µg/mL concentration for all Vibrio isolates (p < 0.0001). Moreover, eradication biofilm assays demonstrated significant eradication against all Vibrio strains at both growth conditions, but biofilm eradication values were substantially lower. Both extract plants demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values at both growth sets, where Eucalyptus extract at 800 µg/mL reduced 70% of biomass and 90% of viable cells for all Vibrio strains (p < 0.0001). Overall results suggested a viable alternative against vibriosis in the shrimp industry in Ecuador.
Assuntos
Antibacterianos , Biofilmes , Eucalyptus , Extratos Vegetais , Psidium , Vibrio cholerae , Vibrio parahaemolyticus , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Psidium/química , Eucalyptus/química , Eucalyptus/microbiologia , Vibrio cholerae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/crescimento & desenvolvimento , Equador , Testes de Sensibilidade Microbiana , Penaeidae/microbiologiaRESUMO
Soils present high fungal diversity, including entomopathogenic species. These fungi are used in pest control, providing easy production, multiplication, application, and dispersion in the field. The objective of the present study was to evaluate entomopathogenic fungal diversity in soils from eucalyptus and soybean crops and natural forest areas. These fungi were isolated using the "Bait Method" with Tenebrio molitor (Linnaeus, 1758) (Coleoptera: Tenebrionidae) larvae from 10 soil samples per area, collected at 10 cm deep in a zig-zag pattern. The isolated entomopathogenic fungi were cultivated in Petri dishes using PDA medium and their mycelia separated after seven days of incubation in a BOD-type chamber. Species of Aspergillus, Beauveria, Cordyceps, Fusarium, Metarhizium, Penicillium and Purpureocillium were identified. The "Bait Method" with T. molitor larvae is efficient to isolate entomopathogenic fungi with higher diversity from soils of the natural forest than the cultivated area.
A diversidade de fungos, incluindo espécies entomopatogênicas, é alta nos solos. Esses fungos são utilizados no manejo de pragas com facilidade de produção, multiplicação, aplicação e dispersão no campo. O objetivo foi avaliar a diversidade de fungos entomopatogênicos em solos de culturas de eucalipto e soja e áreas de mata nativa. Fungos entomopatogênicos foram isolados pelo "Bait Method" com larvas de Tenebrio molitor (Linnaeus, 1758) (Coleoptera: Tenebrionidae) de 10 amostras de solo por área, coletadas a 10 cm de profundidade em zig-zag. Os fungos isolados foram cultivados em três placas de Petri em meio BDA e seus micélios separados após sete dias de incubação em câmara tipo BOD. Fungos dos gêneros Aspergillus, Beauveria, Cordyceps, Fusarium, Metarhizium, Penicillium e Purpureocillium foram identificados. O "Bait Method" com larvas de T. molitor é eficiente para isolar fungos entomopatogênicos com maior diversidade em solos de área de mata nativa que naqueles com culturas de eucalipto e soja.
Assuntos
Microbiologia do Solo , Glycine max/microbiologia , Eucalyptus/microbiologia , Fungos/isolamento & purificação , 24444 , Cordyceps , Beauveria , Metarhizium , FusariumRESUMO
Eucalyptus is the main species for the forestry industry in Brazil. Biotechnology and, more recently, gene editing offer significant opportunities for rapid improvements in Eucalyptus breeding programs. However, the recalcitrance of Eucalyptus species to in vitro culture is also a major limitation for commercial deployment of biotechnology techniques in Eucalyptus improvement. We evaluated various clones of Eucalyptus urophylla for their in vitro regeneration potential identified a clone, BRS07-01, with considerably higher regeneration rate (85%) in organogenesis, and significantly higher than most works described in literature. Endophytic bacteria are widely reported to improve in vitro plant growth and development. Hence, we believe that inclusion of endophytic plant growth promoting bacteria enhanced was responsible for the improved plantlets growth and development of this clone under in vitro culture. Metagenomic analysis was performed to isolate and characterize the prominent endophytic bacteria on BRS07-01 leaf tissue in vitro micro-cultures, and evaluate their impact on plant growth promotion. The analysis revealed the presence of the phyla Firmicutes (35%), Proteobacteria (30%) and much smaller quantities of Actinobacteria, Bacteroidetes, Gemmatimonadetes, Crenarchaeota, Euryarchaeota and Acidobacteria. Of the thirty endophytic bacterial strains isolated, eleven produced indole-3-acetic acid. Two of the isolates were identified as Enterobacter sp. and Paenibacillus polymyxa, which are nitrogen-fixing and capable of phosphate and produce ammonium. These isolates also showed similar positive effects on the germination of common beans (Phaseolus spp.). The isolates will now be tested as a growth promoter in Eucalyptus in vitro cultures. Graphical abstract for the methodology using cultivation independent and dependent methodologies to investigate the endophytic bacteria community from in vitro Eucalyptus urophylla BRS07-01.
Assuntos
Bactérias/isolamento & purificação , Endófitos/isolamento & purificação , Eucalyptus/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Brasil , DNA Bacteriano/genética , DNA Ribossômico/genética , Endófitos/classificação , Endófitos/genética , Endófitos/metabolismo , Eucalyptus/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Metagenômica , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , RNA Ribossômico 16S/genéticaRESUMO
In a first step, essential oils were extracted from Eucalyptus globulus leaves, healthy and with symptoms and signs of Mycosphaerella leaf disease (MLD) and Teratosphaeria leaf disease (TLD), in two leaf stages. Stage 1: sessile, oval leaves covered by a waxy layer of a bluish colour, with opposite phyllotaxis, inserted along stems of quadrangular section. Stage 2: narrow and sickle leaves with a greyish green surface, mainly on the abaxial surface, inserted in alternating pairs along rounded stems. The essential oils were extracted by hydrodistillation and were analyzed by gas chromatography coupled with mass spectrometry. Chemical composition data and percentages of essential oil constituents were submitted to cluster analysis and principal component analysis. In a second step, under in vitro conditions, was evaluated the germination of Teratosphaeria nubilosa (one of the causal agents of TLD) ascospores in contact with the four types of essential oils extracted. The evaluations were performed at 24, 48 and 72 h after the experiments were assembled. The present study made it possible to distinguish and identify the chemical composition of essential oils from the eucalypt leaves used, and allowed 1,8-cineole to be identified as the major component for the essential oils investigated. The contact between essential oils and T. nubilosa spores allowed to prove the inhibition of the ascospores germination, being more efficient for the essential oils extracted from materials with the disease, which presented high amounts of 1,8-cineole.
Assuntos
Ascomicetos , Eucalyptus , Mycosphaerella , Óleos Voláteis , Esporos Fúngicos , Ascomicetos/fisiologia , Eucalyptus/microbiologia , Mycosphaerella/fisiologia , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Folhas de Planta/microbiologia , Esporos Fúngicos/efeitos dos fármacosRESUMO
Eucalyptus grandis is a globally important tree crop. Greenhouse-grown tree seedlings often face water deficit after outplanting to the field, which can affect their survival and establishment severely. This can be alleviated by the application of superabsorbent hydrophilic polymers (SAPs). Growth promoting bacteria can also improve crop abiotic stress tolerance; however, their use in trees is limited, partly due to difficulties in the application and viability loss. In this work, we evaluated the improvement of drought tolerance of E. grandis seedlings by inoculating with two Pseudomonas strains (named M25 and N33), carried by an acrylic-hydrocellulosic SAP. We observed significant bacterial survival in the seedling rhizosphere 50 days after inoculation. Under gradual water deficit conditions, we observed a considerable increase in the water content and wall elasticity of M25-inoculated plants and a trend towards growth promotion with both bacteria. Under rapid water deficit conditions, which caused partial defoliation, both strains significantly enhanced the formation of new leaves, while inoculation with M25 reduced the transpiration rate. Co-inoculation with M25 and N33 substantially increased growth and photosynthetic capacity. We conclude that the selected bacteria can benefit E. grandis early growth and can be easily inoculated at transplant by using an acrylic-hydrocellulosic SAP.
Assuntos
Bactérias/isolamento & purificação , Secas , Eucalyptus/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Polímeros/química , Plântula/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento , Eucalyptus/microbiologia , Raízes de Plantas/microbiologia , Rizosfera , Plântula/microbiologia , ÁguaRESUMO
Three yeast strains were isolated from decaying wood of Chilean Valdivian forest and identified as Meyerozyma guilliermondii, Scheffersomyces coipomensis, and Sugiyamaella paludigena. These strains were able to efficiently grow on the major monomers contained in Pinus spp. and Eucalyptus spp. wood that includes glucose (Glc), xylose (Xyl), and mannose (Man), showing at 28 °C higher uptake rates for Man, and in some cases for Glc, than for Xyl, used as single carbon sources. Nevertheless, in cultures performed on sugar mixtures, the strains displayed a notable preference for Glc. Additionally, in sugar mixtures, the absence of regulatory mechanisms in sugar assimilation (e.g., catabolic repression) was observed and documented when the activities of several enzymes involved in sugar assimilation (i.e., phosphoglucose isomerase, phosphomannose isomerase, and xylulokinase) were determined. The activity of the key enzymes involved in the onset of lipid accumulation (i.e., NAD+-ICDH) and in fatty acid (FA) biosynthesis (i.e., ATP:CL) indicated a significant accumulation of storage lipids (i.e., up to 24%, w/w) containing oleic and palmitic acids as the major components. The present paper is the first report on the potential of M. guilliermondii, S. coipomensis, and S. paludigena as oleaginous yeasts. We conclude that the new isolates, being able to simultaneously assimilate the major lignocellulosic sugars and efficiently convert them into oily biomass, present a biotechnological potential which deserve further investigation.
Assuntos
Florestas , Lignina/metabolismo , Lipídeos/biossíntese , Açúcares/metabolismo , Leveduras/metabolismo , Eucalyptus/microbiologia , Pinus/microbiologia , Madeira/microbiologia , Leveduras/isolamento & purificaçãoRESUMO
Key Message A resistant E. grandis genotype showed a constitutive overexpression of genes related to resistance to myrtle rust caused by A. psidii. Abstract Myrtle rust caused by Austropuccinia psidii is considered one of the most important fungal diseases affecting Eucalyptus spp. plantations in Brazil. Although the selection and planting of resistant eucalypt genotypes have been the major strategies to manage the disease in Brazil, the molecular mechanisms involved in resistance are still unclear. In this study, we evaluated the gene expression profile of two contrasting Eucalyptus grandis genotypes in resistance level to rust by RNA-Seq. The two genotypes showed a very different background gene expression level even without A. psidii infection. The resistant genotype had a constitutive overexpression of a large number of protein-coding genes compared to the susceptible genotype. These genes were mainly associated with signal transduction, photosynthesis, regulation and response to salicylic acid (SA), and protein kinase leucine-rich receptors (PK-LRR). PK-LRR and SA mediated disease resistance are well known to be effective against obligate biotroph pathogens, such as A. psidii. In addition, at 24 h after infection, the susceptible genotype was able to activate some response, however, several resistance-related proteins had their expression level reduced with A. psidii infection. Here, we present the first analysis of E. grandis genotypes transcriptomes infected by A. psidii and it reveals a constitutive overexpression of several resistance-related genes in the resistant genotype compared to the susceptible one. Our findings have the potential to be used as candidate molecular markers for resistance to myrtle rust.
Assuntos
Basidiomycota/patogenicidade , Eucalyptus/genética , Eucalyptus/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Brasil , Resistência à Doença/genética , Eucalyptus/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genótipo , Família Multigênica , Fotossíntese/genética , Doenças das Plantas/genética , Polimorfismo de Nucleotídeo Único , Ácido Salicílico/metabolismoRESUMO
This study aimed to develop and validate a standard area diagram (SAD) set to estimate the severity of bacterial blight of eucalyptus caused by Erwinia psidii. For this purpose, an eight-level SAD was developed and validated by ten inexperienced raters. Accuracy and precision of the estimates by each rater, with and without the SAD, were determined based on Lin's concordance correlation coefficient. The proposed SAD improved the accuracy and precision of the estimates. The SAD set studied here is a useful tool in assessments of bacterial blight of eucalyptus for epidemiological research and breeding programs.(AU)
Este trabalho objetivou o desenvolvimento de uma escala para estimar a severidade da seca-de-ponteiros do eucalipto causada por Erwinia psidii. Para isso, uma escala de oito níveis foi desenvolvida e validada por dez avaliadores inexperientes. A acurácia e precisão das estimativas de cada avaliador, com e sem a escala, foram determinadas baseadas no coeficiente de correlação concordante de Lin. A escala proposta melhorou a acurácia e a precisão das estimativas. A escala estudada se mostrou uma ferramenta útil na avaliação da seca-de-ponteiros do eucalipto para estudos epidemiológicos e em programas de melhoramento.(AU)
Assuntos
Doenças das Plantas/microbiologia , Infecções Bacterianas/classificação , Erwinia , Eucalyptus/microbiologia , Reprodutibilidade dos TestesRESUMO
This study aimed to develop and validate a standard area diagram (SAD) set to estimate the severity of bacterial blight of eucalyptus caused by Erwinia psidii. For this purpose, an eight-level SAD was developed and validated by ten inexperienced raters. Accuracy and precision of the estimates by each rater, with and without the SAD, were determined based on Lin's concordance correlation coefficient. The proposed SAD improved the accuracy and precision of the estimates. The SAD set studied here is a useful tool in assessments of bacterial blight of eucalyptus for epidemiological research and breeding programs.(AU)
Este trabalho objetivou o desenvolvimento de uma escala para estimar a severidade da seca-de-ponteiros do eucalipto causada por Erwinia psidii. Para isso, uma escala de oito níveis foi desenvolvida e validada por dez avaliadores inexperientes. A acurácia e precisão das estimativas de cada avaliador, com e sem a escala, foram determinadas baseadas no coeficiente de correlação concordante de Lin. A escala proposta melhorou a acurácia e a precisão das estimativas. A escala estudada se mostrou uma ferramenta útil na avaliação da seca-de-ponteiros do eucalipto para estudos epidemiológicos e em programas de melhoramento.(AU)
Assuntos
Doenças das Plantas/microbiologia , Infecções Bacterianas/classificação , Erwinia , Eucalyptus/microbiologia , Reprodutibilidade dos TestesRESUMO
To meet a global demand for timber, tree plantations were established in South America during the first half of the 20th century. Extensive plantings of non-native species now are found in Brazil, Chile, Argentina, and Uruguay. In Colombia, miscellaneous plantations were established in the 1950s, during a period of intensive local logging, when policies to limit deforestation in native Quercus humboldtii forests were established. One unforeseen consequence of planting non-native trees was the simultaneous introduction and subsequent persistence of ectomycorrhizal fungi. We sought to document the origins and spread of the introduced Amanita muscaria found in Colombian plantations of the Mexican species Pinus patula, North American species P. taeda, and Australian species Acacia melanoxylon and Eucalyptus globulus. In Colombia, Amanita muscaria is establishing a novel association with native Q. humboldtii and has spread to local Q. humboldtii forests. According to a Bayesian phylogeny and haplotype analysis based on the nuclear rDNA internal transcribed spacer region ITS1-5.8-ITS2 (ITS barcode), A. muscaria individuals found in four exotic plant species, and those colonizing Q. humboldtii roots, have a Eurasian origin and belong to two Eurasian haplotypes. This is the first time the spread of an introduced mutualist fungus into native Colombian Q. humboldtii forests is reported. To arrest its spread, we suggest the use of local inocula made up of native fungi, instead of inocula of introduced fungi.
Assuntos
Amanita/crescimento & desenvolvimento , Amanita/isolamento & purificação , Especificidade de Hospedeiro , Quercus/microbiologia , Acacia/microbiologia , Amanita/genética , Análise por Conglomerados , Colômbia , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Eucalyptus/microbiologia , Florestas , Filogenia , Pinus/microbiologia , RNA Ribossômico 5,8S/genética , Análise de Sequência de DNARESUMO
Eucalyptus plantations offer a cost-effective and renewable source of raw material. There is substantial interest in improving forestry production, especially through sustainable strategies such as the use of plant growth-promoting bacteria. However, little is known about Eucalyptus microbiology. In this study, the endophytic bacterial community was assessed in Eucalyptus urograndis roots using culture-dependent and culture-independent techniques with plants grown under different conditions. Three phyla accounted for approximately 95% of the community, with Actinobacteria corresponding to approximately 59%. This contrasts with previous studies in which Actinobacteria accounted for only 5 to 10%. Our data also revealed a high diversity of bacteria, with 359 different genera but a high level of dominance. Six genera, Mycobacterium, Bradyrhizobium, Streptomyces, Bacillus, Actinospica, and Burkholderia, accounted for more than 50% of the classified sequences. We observed a significant influence of the treatments on some genera, causing changes in the bacterial community structure. The obtained data also suggest that Eucalyptus may benefit from biological nitrogen fixation, with many abundant genera being closely related to nitrogen-fixing bacteria. Using N-depleted media, we also cultured 95 bacterial isolates, of which 24 tested positive for the nifH gene and were able to maintain growth without any N source in the medium.
Assuntos
Bactérias/metabolismo , Endófitos/metabolismo , Eucalyptus/microbiologia , Microbiota , Fixação de Nitrogênio , Raízes de Plantas/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Endófitos/classificação , Endófitos/genética , Endófitos/isolamento & purificação , Filogenia , RNA Ribossômico 16S/genética , Microbiologia do SoloRESUMO
Despite the great advances in chemotherapeutics, infectious diseases are still one of the leading causes of death worldwide. Among some of the clinically relevant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) ranks as one of the most difficult bacteria to treat. It is a common cause of skin, soft-tissue, and endovascular infections, as well as pneumonia, septic arthritis, endocarditis, osteomyelitis, and sepsis. The research on Basidiomycota is extensive; many species show a broad spectrum of pharmacological activities, including antimicrobial activity. The vast majority of the literature to date generally focuses on screening the antibacterial properties of mushroom extracts. A gap still exists in the identification of the individual compounds responsible for these properties, and few low molecular weight compounds have been described. Gymnopilus junonius, the big laughter mushroom, grows wild in Uruguay, especially on Eucalyptus spp. plantations; it is known as the "eucalyptus fungus." In this work, we report the bioguided isolation, structural elucidation, and antistaphylococcal activity of the main antimicrobial components of fresh basidiocarps of G. junonius.
Assuntos
Agaricales/química , Antibacterianos/farmacologia , Misturas Complexas/farmacologia , Carpóforos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Agaricales/crescimento & desenvolvimento , Antibacterianos/isolamento & purificação , Misturas Complexas/isolamento & purificação , Eucalyptus/microbiologia , Testes de Sensibilidade Microbiana , UruguaiRESUMO
Abstract Plant growth-promoting rhizobacteria strains from special formulations have been used to optimize eucalyptus cutting production. To undertake quality control for the formulated products, the rhizobacterial strains should be characterized to assess their purity and authentication. In the present study, we characterized nine strains of rhizobacteria, including three Bacillus subtilis (S1, S2 and 3918), two Pseudomonas sp. (MF4 and FL2), P. putida (MF2), P. fulva (Ca), Frateuria aurantia (R1), and Stenotrophomonas maltophilia (CIIb). The strains were differentiated by colony morphology after 24 h of incubation in three different solid state culture media (glucose-nutritive agar, 523 medium and yeast extract-mannitol agar), sensitivity to a panel of 28 antibiotics (expressed according to the formation of inhibition halos of bacterial growth in the presence of antibiotics), and PCR-RFLP profiles of the 16S rDNA gene produced using nine restriction enzymes. It was possible to differentiate all nine strains of rhizobacteria using their morphological characteristics and sensitivity to antibiotics. The molecular analysis allowed us to separate the strains CIIb, FL2 and R1 from the strains belonging to the genera Bacillus and Pseudomonas. By using these three methods concomitantly, we were able to determine strain purity and perform the authentication.
Assuntos
Bactérias , Eucalyptus/crescimento & desenvolvimento , Eucalyptus/microbiologia , Rizosfera , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Bactérias/genética , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologiaRESUMO
Plant growth-promoting rhizobacteria strains from special formulations have been used to optimize eucalyptus cutting production. To undertake quality control for the formulated products, the rhizobacterial strains should be characterized to assess their purity and authentication. In the present study, we characterized nine strains of rhizobacteria, including three Bacillus subtilis (S1, S2 and 3918), two Pseudomonas sp. (MF4 and FL2), P. putida (MF2), P. fulva (Ca), Frateuria aurantia (R1), and Stenotrophomonas maltophilia (CIIb). The strains were differentiated by colony morphology after 24 h of incubation in three different solid state culture media (glucose-nutritive agar, 523 medium and yeast extract-mannitol agar), sensitivity to a panel of 28 antibiotics (expressed according to the formation of inhibition halos of bacterial growth in the presence of antibiotics), and PCR-RFLP profiles of the 16S rDNA gene produced using nine restriction enzymes. It was possible to differentiate all nine strains of rhizobacteria using their morphological characteristics and sensitivity to antibiotics. The molecular analysis allowed us to separate the strains CIIb, FL2 and R1 from the strains belonging to the genera Bacillus and Pseudomonas. By using these three methods concomitantly, we were able to determine strain purity and perform the authentication.(AU)
Assuntos
Eucalyptus/microbiologia , Bactérias/classificação , Bactérias/imunologia , CrescimentoRESUMO
Plant growth-promoting rhizobacteria strains from special formulations have been used to optimize eucalyptus cutting production. To undertake quality control for the formulated products, the rhizobacterial strains should be characterized to assess their purity and authentication. In the present study, we characterized nine strains of rhizobacteria, including three Bacillus subtilis (S1, S2 and 3918), two Pseudomonas sp. (MF4 and FL2), P. putida (MF2), P. fulva (Ca), Frateuria aurantia (R1), and Stenotrophomonas maltophilia (CIIb). The strains were differentiated by colony morphology after 24h of incubation in three different solid state culture media (glucose-nutritive agar, 523 medium and yeast extract-mannitol agar), sensitivity to a panel of 28 antibiotics (expressed according to the formation of inhibition halos of bacterial growth in the presence of antibiotics), and PCR-RFLP profiles of the 16S rDNA gene produced using nine restriction enzymes. It was possible to differentiate all nine strains of rhizobacteria using their morphological characteristics and sensitivity to antibiotics. The molecular analysis allowed us to separate the strains CIIb, FL2 and R1 from the strains belonging to the genera Bacillus and Pseudomonas. By using these three methods concomitantly, we were able to determine strain purity and perform the authentication.
Assuntos
Bactérias , Eucalyptus/crescimento & desenvolvimento , Eucalyptus/microbiologia , Rizosfera , Antibacterianos/farmacologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genéticaRESUMO
The relationships between plants and endophytic bacteria significantly contribute to plant health and yield. However, the microbial diversity in leaves of Eucalyptus spp. is still poorly characterized. Here, we investigated the endophytic diversity in leaves of hybrid Eucalyptus grandis x E. urophylla (Eucalyptus "urograndis") by using culture-independent and culture-dependent approaches, to better understand their ecology in leaves at different stages of Eucalyptus development, including bacteria with N2 fixation potential. Firmicutes, Proteobacteria (classes alpha-, beta- and gamma-) and Actinobacteria were identified in the Eucalyptus "urograndis" endophytic bacterial community. Within this community, the species Novosphingobium barchaimii, Rhizobium grahamii, Stenotrophomonas panacihumi, Paenibacillus terrigena, P. darwinianus and Terrabacter lapilli represent the first report these bacteria as endophytes. The diversity of the total endophytic bacteria was higher in the leaves from the 'field' (the Shannon-Wiener index, 2.99), followed by the indices obtained in the 'clonal garden' (2.78), the 'recently out from under shade (2.68), 'under shade' (2.63) and 'plants for dispatch' (2.51). In contrast, for diazotrophic bacteria, the highest means of these indices were obtained from the leaves of plants in the 'under shade' (2.56), 'recently out from under shade (2.52)' and 'field' stages (2.54). The distribution of the endophytic bacterial species in Eucalyptus was distinct and specific to the development stages under study, and many of the species had the potential for nitrogen fixation, raising the question of whether these bacteria could contribute to overall nitrogen metabolism of Eucalyptus.
Assuntos
Bactérias/classificação , Endófitos/classificação , Eucalyptus/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , DNA Bacteriano/genética , DNA Ribossômico/genética , Ecologia , Endófitos/genética , Endófitos/isolamento & purificação , Endófitos/fisiologia , Eucalyptus/crescimento & desenvolvimento , Eucalyptus/metabolismo , Fixação de Nitrogênio , Filogenia , Folhas de Planta/microbiologia , RNA Ribossômico 16S/genéticaRESUMO
At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 µM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.
Assuntos
Basidiomycota/fisiologia , Eucalyptus/microbiologia , Manganês/farmacologia , Micorrizas/fisiologia , Basidiomycota/efeitos dos fármacos , Basidiomycota/enzimologia , Basidiomycota/crescimento & desenvolvimento , Catalase/genética , Catalase/metabolismo , Clorofila/fisiologia , Eucalyptus/crescimento & desenvolvimento , Eucalyptus/fisiologia , Fluorescência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Glucose/farmacologia , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Micorrizas/efeitos dos fármacos , Micorrizas/enzimologia , Micorrizas/crescimento & desenvolvimentoRESUMO
Two lanostane triterpenoids (sclerodols A and B) were isolated from the culture of the Eucalyptus grandis derived from the endophyte Scleroderma UFSM Sc1(Persoon) Fries together with three known compounds: one related triterpenoid lanosta-8,23-dien-3ß,25-diol, the disaccharide α,ß-trehalose, and the sugar alcohol mannitol. Their structures were elucidated on the basis of 2D NMR, HRME, and single-crystal X-ray diffraction data. The methanol crude extract and the isolated lanostane triterpenoids showed promising anticandidal activities.
Assuntos
Antifúngicos/química , Basidiomycota/metabolismo , Triterpenos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Cristalografia por Raios X , Eucalyptus/microbiologia , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Conformação Molecular , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
MAIN CONCLUSION: Elicitation of E. grandis plants with Streptomyces PM9 reduced the gray-mold disease, through increasing the levels of enzymes directly related to the induction of plant defense responses, and accumulation of specific phenolic compounds. Members of Eucalyptus are economically important woody species, especially as a raw material in many industrial sectors. Species of this genus are susceptible to pathogens such as Botrytis cinerea (gray mold). Biological control of plant diseases using rhizobacteria is one alternative to reduce the use of pesticides and pathogen attack. This study evaluated the metabolic and phenotypic responses of Eucalyptus grandis and E. globulus plants treated with Streptomyces sp. PM9 and challenged with the pathogenic fungus B. cinerea. Metabolic responses were evaluated by assessing the activities of the enzymes polyphenol oxidase and peroxidase as well as the levels of phenolic compounds and flavonoids. The incidence and progression of the fungal disease in PM9-treated plants and challenged with B. cinerea were evaluated. Treatment with Streptomyces sp. PM9 and challenge with B. cinerea led to changes in the activities of polyphenol oxidase and peroxidase as well as in the levels of phenolic compounds in the plants at different time points. Alterations in enzymes of PM9-treated plants were related to early defense responses in E. grandis. Gallic and chlorogenic acids were on average more abundant, although caffeic acid, benzoic acid and catechin were induced at specific time points during the culture period. Treatment with Streptomyces sp. PM9 significantly delayed the establishment of gray mold in E. grandis plants. These results demonstrate the action of Streptomyces sp. PM9 in inducing plant responses against B. cinerea, making this organism a potential candidate for biological control in Eucalyptus.
Assuntos
Botrytis/patogenicidade , Eucalyptus/fisiologia , Doenças das Plantas/microbiologia , Streptomyces/fisiologia , Catecol Oxidase/metabolismo , Ácido Clorogênico/metabolismo , Eucalyptus/microbiologia , Ácido Gálico/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Hidroxibenzoatos/metabolismo , Peroxidases/metabolismo , Metabolismo SecundárioRESUMO
Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.