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Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.
Assuntos
Bacillus , Proteínas Hemolisinas , Hemólise , Lipopeptídeos , Simulação de Acoplamento Molecular , Staphylococcus aureus , Bacillus/metabolismo , Bacillus/química , Staphylococcus aureus/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Animais , Bovinos , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Mastite Bovina/microbiologia , Mastite Bovina/tratamento farmacológico , Feminino , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Simulação de Dinâmica MolecularRESUMO
Root-knot nematodes (RKN) are one of the most harmful soil-borne plant pathogens in the world. Actinobacteria are known phytopathogen control agents. The aim of this study was to select soil actinobacteria with control potential against the RKN (Meloidogyne javanica) in tomato plants and to determine mechanisms of action. Ten isolates were tested and a significant reduction was observed in the number of M. javanica eggs, and galls 46 days after infestation with the nematode. The results could be explained by the combination of different mechanisms including parasitism and induction of plant defense response. The M. javanica eggs were parasited by all isolates tested. Some isolates reduced the penetration of juveniles into the roots. Other isolates using the split-root method were able to induce systemic defenses in tomato plants. The 4L isolate was selected for analysis of the expression of the plant defense genes TomLoxA, ACCO, PR1, and RBOH1. In plants treated with 4L isolate and M. javanica, there was a significant increase in the number of TomLoxA and ACCO gene transcripts. In plants treated only with M. javanica, only the expression of the RBOH1 and PR1 genes was induced in the first hours after infection. The isolates were identified using 16S rRNA gene sequencing as Streptomyces sp. (1A, 3F, 4L, 6O, 8S, 9T, and 10U), Kribbella sp. (5N), Kitasatospora sp. (2AE), and Lentzea sp. (7P). The efficacy of isolates from the Kitasatospora, Kribbella, and Lentzea genera was reported for the first time, and the efficacy of Streptomyces genus isolates for controlling M. javanica was confirmed. All the isolates tested in this study were efficient against RKN. This study provides the opportunity to investigate bacterial genera that have not yet been explored in the control of M. javanica in tomatoes and other crops.
Assuntos
Actinobacteria , Actinomycetales , Solanum lycopersicum , Tylenchoidea , Animais , Doenças das Plantas/prevenção & controle , Tylenchoidea/genética , Actinobacteria/genética , RNA Ribossômico 16S/genética , Bactérias/genética , Actinomycetales/genética , SoloRESUMO
Fungi belonging to the genus Trichoderma have been widely recognized as efficient controllers of plant diseases. Although the majority of isolates currently deployed, thus far, have been isolated from soil, endophytic Trichoderma spp. is considered to be a promising option for application in biocontrol. In this study, 30 endophytic Trichoderma isolates-obtained from the leaves, stems, and roots of wild Hevea spp. in the Brazilian Amazon-were analyzed using specific DNA barcodes: sequences of internal transcribed spacers 1 and 2 of rDNA (ITS region), genes encoding translation elongation factor 1-α (TEF1-α), and the second largest subunit of RNA polymerase II (RPB2). The genealogical concordance phylogenetic species recognition (GCPSR) concept was used for species delimitation. A phylogenetic analysis showed the occurrence of Trichoderma species, such as T. erinaceum, T. ovalisporum, T. koningiopsis, T. sparsum, T. lentiforme, T. virens, and T. spirale. Molecular and morphological features resulted in the discovery of four new species, such as T. acreanum sp. nov., T. ararianum sp. nov., T. heveae sp. nov., and T. brasiliensis sp. nov. The BI and ML analyses shared a similar topology, providing high support to the final trees. The phylograms show three distinct subclades, namely, T. acreanum and T. ararianum being paraphyletic with T. koningiopsis; T. heveae with T. subviride; and T. brasiliensis with T. brevicompactum. This study adds to our knowledge of the diversity of endophytic Trichoderma species in Neotropical forests and reveals new potential biocontrol agents for the management of plant diseases.
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BACKGROUND: ICEs are mobile genetic elements found integrated into bacterial chromosomes that can excise and be transferred to a new cell. They play an important role in horizontal gene transmission and carry accessory genes that may provide interesting phenotypes for the bacteria. Here, we seek to research the presence and the role of ICEs in 300 genomes of phytopathogenic bacteria with the greatest scientific and economic impact. RESULTS: Seventy-eight ICEs (45 distinct elements) were identified and characterized in chromosomes of Agrobacterium tumefaciens, Dickeya dadantii, and D. solani, Pectobacterium carotovorum and P. atrosepticum, Pseudomonas syringae, Ralstonia solanacearum Species Complex, and Xanthomonas campestris. Intriguingly, the co-occurrence of four ICEs was observed in some P. syringae strains. Moreover, we identified 31 novel elements, carrying 396 accessory genes with potential influence on virulence and fitness, such as genes coding for functions related to T3SS, cell wall degradation and resistance to heavy metals. We also present the analysis of previously reported data on the expression of cargo genes related to the virulence of P. atrosepticum ICEs, which evidences the role of these genes in the infection process of tobacco plants. CONCLUSIONS: Altogether, this paper has highlighted the potential of ICEs to affect the pathogenicity and lifestyle of these phytopathogens and direct the spread of significant putative virulence genes in phytopathogenic bacteria.
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Anthracnose, caused by Colletotrichum lindemuthianum, is a disease affecting the common bean plant, Phaseolus vulgaris. To establish infection, the phytopathogen must survive the toxic compounds (phytoanticipins and phytoalexins) that are produced by the plant as a defense mechanism. To study the detoxification and efflux mechanisms in C. lindemuthianum, the abcCl1 gene, which encodes an ABC transporter, was analyzed. The abcCl1 gene (4558 pb) was predicted to encode a 1450-amino acid protein. Structural analysis of 11 genome sequences from Colletotrichum spp. showed that the number of ABC transporters varied from 34 to 64. AbcCl1 was classified in the ABC-G family of transporters, and it appears to be orthologs to ABC1 from Magnaporthe grisea and FcABC1 from Fusarium culmorum, which are involved in pleiotropic drug resistance. A abcT3 (ΔabcCl1) strain showed reduction on aggressivity when inoculated on bean leaves that presented diminishing anthracnose symptoms, which suggests the important role of AbcCl1 as a virulence factor and in fungal resistance to host compounds. The expression of abcCl1 increased in response to different toxic compounds, such as eugenol, hygromycin, and pisatin phytoalexin. Together, these results suggest that AbcCl1 is involved in fungal resistance to the toxic compounds produced by plants or antagonistic microorganisms.
Assuntos
Colletotrichum , Phaseolus , Colletotrichum/genética , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Fatores de Virulência/genéticaRESUMO
AIM: To identify and analyse genes that encode pectinases in the genome of the fungus Colletotrichum lindemuthianum, evaluate the expression of these genes, and compare putative pectinases found in C. lindemuthianum with pectinases produced by other fungi and oomycetes with different lifestyles. METHODS AND RESULTS: Genes encoding pectinases in the genome of C. lindemuthianum were identified and analysed. The expression of these genes was analysed. Pectinases from C. lindemuthianum were compared with pectinases from other fungi that have different lifestyles, and the pectinase activity in some of these fungi was quantified. Fifty-eight genes encoding pectinases were identified in C. lindemuthianum. At least six types of enzymes involved in pectin degradation were identified, with pectate lyases and polygalacturonases being the most abundant. Twenty-seven genes encoding pectinases were differentially expressed at some point in C. lindemuthianum during their interactions with their host. For each type of pectinase, there were at least three isoenzyme groups. The number of pectinases present in fungi with different lifestyles seemed to be related more to the lifestyle than to the taxonomic relationship between them. Only phytopathogenic fungi showed pectate lyase activity. CONCLUSIONS: The collective results demonstrate the pectinolytic arsenal of C. lindemuthianum, with many and diverse genes encoding pectinases more than that found in other phytopathogens, which suggests that at least part of these pectinases must be important for the pathogenicity of the fungus C. lindemuthianum. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of these pectinases could further the understanding of the importance of this broad pectinolytic arsenal in the common bean infection and could be exploited for biotechnological purposes.
Assuntos
Colletotrichum , Fabaceae , Colletotrichum/genética , Fabaceae/microbiologia , Fungos/metabolismo , Poligalacturonase/genética , Poligalacturonase/metabolismoRESUMO
The Núcleo de Estudos em Microbiologia Agrícola (NEMA) is an academic-scientific group created by graduate students in the Post Graduate in Agricultural Microbiology in the Department of Microbiology at Universidade Federal de Viçosa, Brazil. NEMA's purposes include promoting and sharing research and knowledge on microbiology in different fields of application. Here, we will comment on our experience in organizing the Summer School on Microbiology and teaching microbiology to undergraduate students during the program. NEMA offers this annual event to disseminate and stimulate knowledge about microbiology for undergraduate students in a participatory, collaborative and interactive way.
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Microbiologia/educação , Treinamento por Simulação/organização & administração , Ensino/normas , Brasil , Feminino , Humanos , Masculino , Treinamento por Simulação/normas , Universidades , Adulto JovemRESUMO
Ralstonia solanacearum, a soil-borne plant pathogen, encompasses a large number of strains known as R. solanacearum species complex (RSSC). Although it has been suggested that mobile genetic elements (MGEs) may play an important role in the RSSC genome, the evolutionary impact of these elements remains unknown. Here, we identified and analysed Integrative and Conjugative Elements (ICEs) and Genomic Islands (GIs) in the 121 genomes published for Ralstonia spp., including RSSC strains and three other non-plant pathogenic Ralstonia spp. Our results provided a dataset of 12 ICEs and 31 GIs distributed throughout Ralstonia spp. Four novel ICEs in RSSC were found. Some of these elements cover 5% of the host genome and carry accessory genes with a potential impact on the fitness and pathogenicity of RSSC. In addition, phylogenetic analysis revealed that these MGEs clustered to the same species, but there is evidence of strains from different countries that host the same element. Our results provide novel insight into the RSSC adaptation, opening new paths to a better understanding of how these elements affect this soil-borne plant pathogen.
Assuntos
Evolução Biológica , Conjugação Genética/genética , Ilhas Genômicas/genética , Ralstonia solanacearum/genética , Genoma Bacteriano , Funções Verossimilhança , Fases de Leitura Aberta/genética , FilogeniaRESUMO
In this study, five bacteriocin-producing Lactococcus lactis strains were identified from different naturally fermented Brazilian sausages. Ion exchange and reversed-phase chromatographies were used to purify the bacteriocins from culture supernatant of the five strains. Mass spectrometry (MALDI-TOF/TOF) showed that the molecular masses of the bactericoins from L. lactis ID1.5, ID3.1, ID8.5, PD4.7, and PR3.1 were 3330.567 Da, 3330.514 Da, 3329.985 Da, 3329.561 Da, and 3329.591 Da, respectively. PCR product sequence analysis confirmed that the structural genes of bacteriocins produced by the five isolates are identical to the lantibiotic nisin Z. Optimal nisin Z production was achieved in tryptone and casein peptone, at pH 6.0 or 6.5. The most favorable temperatures for nisin Z production were 25°C and 30°C, and its production was better under aerobic than anaerobic condition. The type of carbon source appeared to be an important factor for nisin Z production. While sucrose was found to be the most efficient carbon source for nisin Z production by four L. lactis isolates, fructose was the best for one isolate. Lactose was also a good energy source for nisin Z production. Surprisingly, glucose was clearly the poorest carbon source for nisin Z production. The five isolates produced different amounts of the bacteriocin, L. lactis ID1.5 and ID8.5 isolates being the best nisin Z producers. DNA sequence analysis did not reveal any sequence differences in the nisZ and nisF promoter regions that could explain the differences in nisin Z production, suggesting that there should be other factors responsible for differential nisin Z production by the isolates.
RESUMO
The genus Colletotrichum comprises species with different lifestyles but is mainly known for phytopathogenic species that infect crops of agronomic relevance causing considerable losses. The fungi of the genus Colletotrichum are distributed in species complexes and within each complex some species have particularities regarding their lifestyle. The most commonly found and described lifestyles in Colletotrichum are endophytic and hemibiotrophic phytopathogenic. Several of these phytopathogenic species show wide genetic variability, which makes long-term maintenance of resistance in plants difficult. Different mechanisms may play an important role in the emergence of genetic variants but are not yet fully understood in this genus. These mechanisms include heterokaryosis, a parasexual cycle, sexual cycle, transposable element activity, and repeat-induced point mutations. This review provides an overview of the genus Colletotrichum, the species complexes described so far and the most common lifestyles in the genus, with a special emphasis on the mechanisms that may be responsible, at least in part, for the emergence of new genotypes under field conditions.
Assuntos
Colletotrichum/crescimento & desenvolvimento , Colletotrichum/genética , Variação Genética/genética , Doenças das Plantas/microbiologia , Elementos de DNA Transponíveis/genética , Endófitos , Genoma Fúngico/genética , Especificidade de Hospedeiro , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade da EspécieRESUMO
Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.
Assuntos
Colletotrichum/enzimologia , Colletotrichum/patogenicidade , Histidina Quinase/genética , Phaseolus/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Sequência de Aminoácidos , Colletotrichum/genética , Enzimas de Restrição do DNA/metabolismo , Histidina Quinase/metabolismo , Mutagênese Insercional , Phaseolus/microbiologia , Doenças das Plantas/terapia , Folhas de Planta/microbiologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean (Phaseolus vulgaris), and anthracnose is one of the most devastating diseases of this plant species. However, little is known about the proteins that are essential for the fungus-plant interactions. Knowledge of the fungus' arsenal of effector proteins is of great importance for understanding this pathosystem. In this work, we analyzed for the first time the arsenal of Colletotrichum lindemuthianum effector candidates (ClECs) and compared them with effector proteins from other species of the genus Colletotrichum, providing a valuable resource for studying the infection mechanisms of these pathogens in their hosts. Isolates of two physiological races (83.501 and 89 A2 2-3) of C. lindemuthianum were used to predict 353 and 349 ClECs, respectively. Of these ClECs, 63% were found to be rich in cysteine, have repetitive sequences of amino acids, and/or possess nuclear localization sequences. Several conserved domains were found between the ClECs. We also applied the effector prediction to nine species in the genus Colletotrichum, and the results ranged from 247 predicted effectors in Colletotrichum graminicola to 446 in Colletotrichum orbiculare. Twelve conserved domains were predicted in the effector candidates of all analyzed species of Colletotrichum. An expression analysis of the eight genes encoding the effector candidates in C. lindemuthianum revealed their induction during the biotrophic phase of the fungus on the bean.
Assuntos
Colletotrichum/genética , Colletotrichum/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Sequência de Aminoácidos/genética , Sequência de Bases , Colletotrichum/isolamento & purificação , Expressão Gênica/genética , Perfilação da Expressão Gênica , Domínios Proteicos/genética , Análise de Sequência de DNARESUMO
One of the major challenges of agriculture currently is to obtain higher crop yield. Environmental conditions, cultivar quality, and plant diseases greatly affect plant productivity. On the other hand, several endophytic Bacillus species have emerged as a complementary, efficient, and safe alternative to current crop management practices. The ability of Bacillus species to form spores, which resist adverse conditions, is an advantage of the genus for use in formulations. Endophytic Bacillus species provide plants with a wide range of benefits, including protection against phytopathogenic microorganisms, insects, and nematodes, eliciting resistance, and promoting plant growth, without causing damage to the environment. Bacillus thuringiensis, B. subtilis, B. amyloliquefaciens, B. velezensis, B. cereus, B. pumilus, and B. licheniformis are the most studied Bacillus species for application in agriculture, although other species within the genus have also shown great potential. Due to the increasing number of whole-genome sequenced endophytic Bacillus spp. strains, various bioactive compounds have been predicted. These data reveal endophytic Bacillus species as an underexploited source of novel molecules of biotechnological interest. In this review, we discuss how endophytic Bacillus species are a valuable multifunctional toolbox to be integrated with crop management practices for achieving higher crop yield.
Assuntos
Bacillus/fisiologia , Endófitos/fisiologia , Plantas/microbiologia , Anti-Infecciosos/metabolismo , Bacillus/classificação , Bacillus/genética , Bactérias/patogenicidade , Agentes de Controle Biológico , Biotecnologia , Produção Agrícola , Produtos Agrícolas , Endófitos/genética , Genoma Bacteriano , Controle Biológico de Vetores , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Reguladores de Crescimento de Plantas , Percepção de Quorum , Microbiologia do Solo , Sequenciamento Completo do GenomaRESUMO
Fungi of the genus Colletotrichum are economically important and are used as models in plant-pathogen interaction studies. In this study, the complete mitochondrial genomes of two Colletotrichum lindemuthianum isolates were sequenced and compared with the mitochondrial genomes of seven species of Colletotrichum. The mitochondrial genome of C. lindemuthianum is a typical circular molecule 37,446 bp (isolate 89 A2 2-3) and 37,440 bp (isolate 83.501) in length. The difference of six nucleotides between the two genomes is the result of a deletion in the ribosomal protein S3 (rps3) gene in the 83.501 isolate. In addition, substitution of adenine for guanine within the rps3 gene in the mitochondrial genome of the 83.501 isolate was observed. Compared to the previously sequenced C. lindemuthianum mitochondrial genome, an exon no annotated in the cytochrome c oxidase I (cox1) gene and a non-conserved open reading frame (ncORF) were observed. The size of the mitochondrial genomes of the seven species of Colletotrichum was highly variable, being attributed mainly to the ncORF, ranging from one to 10 and also from introns ranging from one to 11 and which encode a total of up to nine homing endonucleases. This paper reports for the first time by means of transcriptome that then ncORFs are transcribed in Colletotrichum spp. Phylogeny data revealed that core mitochondrial genes could be used as an alternative in phylogenetic relationship studies in Colletotrichum spp. This work contributes to the genetic and biological knowledge of Colletotrichum spp., which is of great economic and scientific importance.
Assuntos
Colletotrichum/genética , Genoma Mitocondrial , Colletotrichum/isolamento & purificação , DNA Circular/química , DNA Circular/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , Éxons , Genes Mitocondriais , Tamanho do Genoma , Fases de Leitura Aberta , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Mutação Puntual , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Novel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID1.5 were isolated and partially characterized. Following purification by cationic exchange and a solid-phase C18 column, antimicrobial activity was recovered after three runs of RPC using 60% (v/v) and 100% (v/v) of 2-propanol for elution, suggesting that more than one antimicrobial compound were produced by L. lactis ID1.5, which were in this study called compounds AI and AII. The mass spectrum of AI and AII showed major intensity ions at m/z 1070.05 and 955.9 Da, respectively. The compound AI showed a spectrum of antimicrobial activity mainly against L. lactis species, while the organisms most sensitive to compound AII were Bacillus subtilis, Listeria innocua, Streptococcus pneumoniae and Pseudomonas aeruginosa. The antimicrobial activity of both compounds was suppressed by treatment with Tween 80. Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L. lactis, and have a significant inhibitory effect against two clinically important respiratory pathogens.
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Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Lactococcus lactis/química , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Bacteriocinas/química , Bacteriocinas/metabolismo , Microbiologia de Alimentos , Lactobacillaceae/efeitos dos fármacos , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/metabolismo , Listeria/efeitos dos fármacos , Polissorbatos , Pseudomonas aeruginosa/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Colletotrichum lindemuthianum is the causal agent of anthracnose in common beans, one of the main limiting factors of their culture. Here, we report for the first time, to our knowledge, a draft of the complete genome sequences of two isolates belonging to 83.501 and 89 A2 2-3 of C. lindemutuianum.
RESUMO
Endophytic bacteria play a key role in the biocontrol of phytopathogenic microorganisms. In this study, genotypic diversity was analyzed via repetitive element PCR (rep-PCR) of endophytic isolates of the phylum Actinobacteria that were previously collected from leaves of cultivars of common bean (Phaseolus vulgaris). Considerable variability was observed, which has not been reported previously for this phylum of endophytic bacteria of the common bean. Furthermore, the ethanol extracts from cultures of various isolates inhibited the growth of pathogenic bacteria in vitro, especially Gram-positive pathogens. Extracts from cultures of Microbacterium testaceum BAC1065 and BAC1093, which were both isolated from the 'Talismã' cultivar, strongly inhibited most of the pathogenic bacteria tested. Bean endophytic bacteria were also demonstrated to have the potential to inhibit the quorum sensing of Gram-negative bacteria. This mechanism may regulate the production of virulence factors in pathogens. The ability to inhibit quorum sensing has also not been reported previously for endophytic microorganisms of P. vulgaris. Furthermore, M. testaceum with capacity to inhibit quorum sensing appears to be widespread in common bean. The genomic profiles of M. testaceum were also analyzed via pulsed-field gel electrophoresis, and greater differentiation was observed using this method than rep-PCR; in general, no groups were formed based on the cultivar of origin. This study showed for the first time that endophytic bacteria from common bean plants exhibit high variability and may be useful for the development of strategies for the biological control of diseases in this important legume plant.
Assuntos
Actinobacteria/classificação , Actinobacteria/fisiologia , Antibacterianos/isolamento & purificação , Endófitos/classificação , Endófitos/fisiologia , Phaseolus/microbiologia , Percepção de Quorum , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Antibacterianos/farmacologia , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Endófitos/genética , Endófitos/isolamento & purificação , Variação Genética , Tipagem Molecular , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Endophytic fungi are microorganisms that live within plant tissues without causing disease during part of their life cycle. With the isolation and identification of these fungi, new species are being discovered, and ecological relationships with their hosts have also been studied. In Glycine max, limited studies have investigated the isolation and distribution of endophytic fungi throughout leaves and roots. The distribution of these fungi in various plant organs differs in diversity and abundance, even when analyzed using molecular techniques that can evaluate fungal communities in different parts of the plants, such as denaturing gradient gel electrophoresis (DGGE). Our results show there is greater species richness of culturable endophytic filamentous fungi in the leaves G. max as compared to roots. Additionally, the leaves had high values for diversity indices, i.e. Simpsons, Shannon and Equitability. Conversely, dominance index was higher in roots as compared to leaves. The fungi Ampelomyces sp., Cladosporium cladosporioides, Colletotrichum gloeosporioides, Diaporthe helianthi, Guignardia mangiferae and Phoma sp. were more frequently isolated from the leaves, whereas the fungi Fusarium oxysporum, Fusarium solani and Fusarium sp. were prevalent in the roots. However, by evaluating the two communities by DGGE, we concluded that the species richness was higher in the roots than in the leaves. UPGMA analysis showed consistent clustering of isolates; however, the fungus Leptospora rubella, which belongs to the order Dothideales, was grouped among species of the order Pleosporales. The presence of endophytic Fusarium species in G. max roots is unsurprising, since Fusarium spp. isolates have been previously described as endophyte in other reports. However, it remains to be determined whether the G. max Fusarium endophytes are latent pathogens or non-pathogenic forms that benefit the plant. This study provides a broader knowledge of the distribution of the fungal community in G. max leaves and roots, and identifies the genetic relationships among the isolated species.
Assuntos
Endófitos/classificação , Fungos/classificação , Fungos/isolamento & purificação , Glycine max/microbiologia , Sequência de Bases , Biodiversidade , Brasil , DNA Ribossômico , Eletroforese em Gel de Gradiente Desnaturante/métodos , Endófitos/isolamento & purificação , Fungos/patogenicidade , Dados de Sequência Molecular , Filogenia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologiaRESUMO
The phy gene, which encodes a phytase in Penicillium chrysogenum CCT 1273, was cloned into the vector pAN-52-1-phy and the resulting plasmid was used for the cotransformation of Penicillium griseoroseum PG63 protoplasts. Among the 91 transformants obtained, 23 were cotransformants. From there, the phytase activity of these 23 transformants was evaluated and P. griseoroseum T73 showed the highest. The recombinant strain P. griseoroseum T73 contained the phy gene integrated in at least three sites of the genome and showed a 5.1-fold increase in phytase activity in comparison to the host strain (from 0.56 ± 0.2 to 2.86 ± 0.4 U µg protein(-1)). The deduced PHY protein has 483 amino acids; an isoelectric point (pI) higher than that reported for phytases from filamentous fungi (7.6); higher activity at pH 2.0 (73%), pH 5.0 (100%) and 50 °C; and is stable at pH values 3.0-8.0 and temperatures 70-80 °C. PHY produced by the recombinant strain P. griseoroseum T73 was stable after four weeks of storage at -20, 8 and 25 °C and was effective in releasing Pi, especially from soybeans. The data presented here show that P. griseoroseum is a successful host for expression of heterologous protein and suggest the potential use of PHY in the animal nutrition industry.
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Clonagem Molecular , Expressão Gênica , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , 6-Fitase/química , Sequência de Aminoácidos , Sequência de Bases , Ativação Enzimática , Dosagem de Genes , Dados de Sequência Molecular , Proteínas Recombinantes , Alinhamento de Sequência , TermodinâmicaRESUMO
The fungus Penicillium griseoroseum has the potential for application on an industrial scale as a host for the production of homologous and heterologous proteins, mainly because it does not produce some mycotoxins or secrete proteases under the growth conditions for pectinase production. However, for the fungus to be used effectively as an expression heterologous system, an understanding of the organization of its genome, as well as the mechanisms of gene expression and protein production, is required. In the present study, the size of the P. griseoroseum genome was estimated to be 29.8-31.5 Mb, distributed among four chromosomes. An analysis of plg1 and pgg2 pectinolytic genes expression and copy number in recombinant multi-copy strains of P. griseoroseum demonstrated that an increase in the number of gene copies could increase enzyme production, but the transcription could be affected by the gene integration position. Placing a copy of the plg1 gene under the control of the gpd promoter of Aspergillus nidulans yielded a 200-fold increase in transcription levels compared to the endogenous gene, and two copies of the pgg2 gene produced an 1100-fold increase compared with the endogenous gene. These results demonstrated that transcription, translation, and protein secretion in the fungus P. griseoroseum respond to an increased number of gene copies in the genome. The processing capacity and efficiency of protein secretion in P. griseoroseum are consistent with our premise that this fungus can be used for the industrial-scale production of several enzymes.