Investigating the impact of insertion sequences and transposons in the genomes of the most significant phytopathogenic bacteria.

Genetic variability in phytopathogens is one of the main problems encountered for effective plant disease control. This fact may be related to the presence of transposable elements (TEs), but little is known about their role in host genomes. Here, we performed the most comprehensive analysis of insertion sequences (ISs) and transposons (Tns) in the genomes of the most important bacterial plant pathogens. A total of 35 692 ISs and 71 transposons were identified in 270 complete genomes. The level of pathogen-host specialization was found to be a significant determinant of the element distribution among the species. Some Tns were identified as carrying virulence factors, such as genes encoding effector proteins of the type III secretion system and resistance genes for the antimicrobial streptomycin. Evidence for IS-mediated ectopic recombination was identified in Xanthomonas genomes. Moreover, we found that IS elements tend to be inserted in regions near virulence and fitness genes, such ISs disrupting avirulence genes in X. oryzae genomes. In addition, transcriptome analysis under different stress conditions revealed differences in the expression of genes encoding transposases in the Ralstonia solanacearum, X. oryzae, and P. syringae species. Lastly, we also investigated the role of Tns in regulation via small noncoding regulatory RNAs and found these elements may target plant-cell transcriptional activators. Taken together, the results indicate that TEs may have a fundamental role in variability and virulence in plant pathogenic bacteria.

Transposable elements contribute to the genome plasticity of Ralstonia solanacearum species complex.

The extensive genetic diversity of Ralstonia solanacearum, a serious soil-borne phytopathogen, has led to the concept that R. solanacearum encompasses a species complex [R. solanacearum species complex (RSSC)]. Insertion sequences (ISs) are suggested to play an important role in the genome evolution of this pathogen. Here, we identified and analysed transposable elements (TEs), ISs and transposons, in 106 RSSC genomes and 15 Ralstonia spp. We mapped 10 259 IS elements in the complete genome of 62 representative RSSC strains and closely related Ralstonia spp. A unique set of 20 IS families was widespread across the strains, IS5 and IS3 being the most abundant. Our results showed six novel transposon sequences belonging to the Tn3 family carrying passenger genes encoding antibiotic resistance and avirulence proteins. In addition, internal rearrangement events associated with ISs were demonstrated in Ralstonia pseudosolanacearum strains. We also mapped IS elements interrupting avirulence genes, which provided evidence that ISs plays an important role in virulence evolution of RSSC. Additionally, the activity of ISs was demonstrated by transcriptome analysis and DNA hybridization in R. solanacearum isolates. Altogether, we have provided collective data of TEs in RSSC genomes, opening a new path for understanding their evolutionary impact on the genome evolution and diversity of this important plant pathogen.

Enzyme Production by Induratia spp. Isolated from Coffee Plants in Brazil

Abstract Endophytic fungi belonging to the genus Muscodor now transferred to Induratia are known producers of bioactive volatile organic compounds (VOCs) with many industrial applications. However, the members of this genus have rarely been reported to produce non-volatile metabolites including enzyme. Enzymes of the endophytes are degraders of the polysaccharides available in the host plants and the knowledge of enzyme production by Induratia spp. may provide insights into their possible biotechnological applications. The aim of this study was to evaluate the activity of amylase, cellulase, lipase, pectinase, phytase, protease, endo β-1,4 glucanase and exo β-1,4 glucanase enzymes produced by fungi of the species Induratia coffeana, Induratia yucatanensis and Induratia sp. isolated from organic coffee plants. All Induratia spp. were able to produce the extracellular enzymes cellulase, pectinase, protease, and phytase. Eight fungi were able to produce lipase and four produced amylase. The specific activity of endo β-1, 4 glucanase and exo β-1,4 glucanase enzymes were detected for 9 and 8 endophytic fungi, respectively. This work demonstrated for the first time, the array of enzymes produced by Induratia spp. isolated from Coffea arabica in organic systems in Brazil.

Genome analysis reveals insights of the endophytic Bacillus toyonensis BAC3151 as a potentially novel agent for biocontrol of plant pathogens.

Diseases caused by phytopathogenic microorganisms account for enormous losses for agribusiness. Although Bacillus species are recognized as being antimicrobial producers and some may provide benefits to plants, the association between Bacillus toyonensis and plants has not been studied. In this study, the whole-genome sequenced endophytic B. toyonensis BAC3151, which has demonstrated antimicrobial activity and quorum sensing inhibition of phytopathogenic bacteria, was investigated for its potential for the production of compounds for biocontrol of plant pathogens. Four whole-genome sequenced B. toyonensis strains shared 3811 protein-coding DNA sequences (CDSs), while strain-specific CDSs, such as biosynthetic gene clusters of antimicrobials, were associated with specific chromosomal regions and mobile genetic elements of the strains. B. toyonensis strains had a higher frequency of putative bacteriocins gene clusters than that of Bacillus species traditionally used for the production of antimicrobials. In addition, gene clusters potentially involved in the production of novel bacteriocins were found in BAC3151, as well as biosynthetic genes of several other compounds, including non-ribosomal peptides, N-acyl homoserine lactonase and chitinases, revealing a genetic repertoire for antimicrobial synthesis greater than that of other Bacillus strains that have demonstrated effective activity against phytopathogens. This study showed for the first time that B. toyonensis has potential to produce various antimicrobials, and the analyses performed indicated that the endophytic strain BAC3151 can be useful for the development of new strategies to control microbial diseases in plants that are responsible for large damages in agricultural crops.

Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Antimicrobial activity of endophytic fungi from coffee plants / Atividade antimicrobiana de fungos endofíticos de plantas de café

Endophytic fungi are a promising source for discovery of compounds with biotechnological potential. The aim of this study was to select and identify endophytic fungi from Coffea arabica that produce volatile organic compounds (VOCs), evaluate the effect of the VOCs produced by endophytic fungi on the growth of Rhizoctonia solani, Fusarium oxysporum, Phoma sp., Botrytis cinerea, Fusarium solani, Fusarium verticillioides, Cercospora coffeicola and Pestalotia longisetula, and select endophytic fungi with potential for biological control of Aspergillus ochraceus inoculated in coffee beans and F. verticillioides inoculated in corn seeds. An isolate of Muscodor albus was used as selection tool for VOC producing fungi. Among the 400 endophytic fungi isolates, 11 were able to grow in the presence of VOCs produced by M. albus. These fungi were identified as Muscodor spp. (9) and Simplicillium sp. according to searches in UNITE database using DNA sequences of internal transcribed spacer (ITS). The VOC's produced by endophytic fungi inhibited the growth the phytopathogenic fungi with different efficacies, compared to the control. The VOCs produced by Muscodor coffeanum (COAD 1842) showed fungicidal effect against A. ochraceus on coffee beans. Six endophytic fungi completely inhibited growth of F. verticillioides inoculated in corn seeds. This study demonstrates that the volatile-compound producing endophytic fungi, isolated from Coffea arabica, are promising sources of bioactive compounds.

Fungos endofíticos são uma fonte promissora para a descoberta de compostos com potencial biotecnológico. O objetivo deste estudo foi selecionar e identificar fungos endofíticos de Coffea arabica que produzem compostos orgânicos voláteis (COVs), avaliar o efeito dos compostos orgânicos voláteis produzido por fungos endofíticos sobre o crescimento de Rhizoctonia solani, Fusarium oxysporum, Phoma sp., Botrytis cinerea, Fusarium solani, Fusarium verticillioides, Cercospora coffeicola e Pestalotia longisetula e selecionar fungos endofíticos com potencial para controle biológico de Aspergillus ochraceus inoculado em grãos de café e F. verticillioides inoculado em sementes de milho. Um isolado de Muscodor albus foi utilizado como ferramenta de seleção para fungos endofíticos produtores de COVs. Dentre os 400 fungos endofíticos isolados, 11 foram capazes de crescer na presença de COVs produzidos por M. albus. Estes fungos foram identificados como Muscodor spp. (9) e Simplicillium sp. de acordo com pesquisas na base de dados UNITE usando sequências de DNA do espaçador transcrito interno (ITS). Os COVs produzidos por fungos endofíticos inibiram o crescimento dos fungos fitopatogênicos em comparação com o controle com diferentes eficácias. Os COVs produzidos por Muscodor coffeanum (COAD 1842) apresentou efeito fungicida contra A. ochraceus em grãos de café. Seis fungos endofíticos inibiram completamente o crescimento de F. verticillioides inoculado em sementes de milho. Este estudo demonstra que os fungos endofíticos produtores de compostos voláteis isolados de Coffea arabica são fontes promissoras de compostos bioativos.

Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression.

Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Evidence of ectopic recombination and a repeat-induced point (RIP) mutation in the genome of Sclerotinia sclerotiorum, the agent responsible for white mold.

Two retrotransposons from the superfamilies Copia and Gypsy named as Copia-LTR_SS and Gypsy-LTR_SS, respectively, were identified in the genomic bank of Sclerotinia sclerotiorum. These transposable elements (TEs) contained direct and preserved long terminal repeats (LTR). Domains related to codified regions for gag protein, integrase, reverse transcriptase and RNAse H were identified in Copia-LTR_SS, whereas in Gypsy-LTR_SS only domains for gag, reverse transcriptase and RNAse H were found. The abundance of identified LTR-Solo suggested possible genetic recombination events in the S. sclerotiorum genome. Furthermore, alignment of the sequences for LTR elements from each superfamily suggested the presence of a RIP (repeat-induced point mutation) silencing mechanism that may directly affect the evolution of this species.

Isolation and characterization of endophytic bacteria isolated from the leaves of the common bean (Phaseolus vulgaris)

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris. leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10² to 2.8 x 10³ CFU g-1 of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.

Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp

The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 10(6) protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 percent); 80.0 percent of protoplasts were nucleated, and 20.0 percent anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 10(7) protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 percent using 0.5 M sucrose as osmotic stabilizer; 88.8 percent of protoplasts were nucleated, and 11.2 percent anucleated.

O objetivo deste trabalho foi padronizar as condições de obtenção e regeneração de protoplastos de Epulorhiza repens e Ceratorhiza sp. Para o fungo E. repens, a maior produção de protoplastos, 8.0 x 10(6) protoplastos/mL, foi obtida em KCl 0.6 M, na presença de 15 mg/mL de "Lysing Enzymes" e micélio fúngico com 2 dias de idade. A maior freqüência de regeneração obtida foi de 8,5 por cento quando sacarose 0.5 M foi utilizada como estabilizador osmótico. Do total de protoplastos obtidos, 80 por cento eram nucleados e 20 por cento anucleados. Para Ceratorhiza sp., a maior produção de protoplastos, 4,0 x 10(7) protoplastos/mL, foi obtida em NaCl 0.6 M, na presença de 15 mg/mL de "Lysing Enzymes" e 15mg/mL de Glucanex, e micélio fúngico com 2 dias de idade. A maior freqüência de regeneração obtida foi de 6.7 por cento utilizando sacarose 0.5 M como estabilizador osmótico. Do total de protoplastos obtidos, 88.8 por cento eram nucleados e 1.2 por cento anucleados. O estabelecimento de protocolo otimizado para obtenção e regeneração de protoplastos dos fungos E. repens e Ceratorhiza sp. é importante, permitindo o estabelecimento de técnicas de transformação genética, o isolamento de mutantes, a determinação de cariótipo eletroforético e o cruzamento de linhagens.

Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao

The presence of restriction enzymes in the transformation mixture improved the efficiency of transformation in Moniliophthora perniciosa. The influence of the vector shape (linear or circular), the patterns of plasmid integration in genomic sites and the influence of the promoter used to express the gene marker were also analyzed. The addition of BamHI or NotI increased the number of transformants by 3-10-fold and 3-fold, respectively, over the control without added enzyme. The use of pre-linearized plasmid did not increase the transformation efficiency in comparison with the circular plasmid. However, the frequency of multi-copy transformants increased significantly. The transformation procedure here reported resulted in better production of protoplasts and transformation efficiency. In addition, the time necessary for the detection of the first transformants and the number of insertions were reduced.

A presença de enzima de restrição na mistura de transformação aumentou a eficiência da transformação em Moniliophthora perniciosa. A influência da forma do vetor (linear ou circular), o padrão de integração do plasmídeo nos sítios genômicos e a influência do promotor usado para expressar o gene marcador foram também analisados. A adição de BamHI ou NotI aumentou o número de transformantes 3-10 vezes e 3 vezes, respectivamente, em relação ao controle sem a adição da enzima. O uso de plasmídeos pré-linearizados não aumentou a eficiência da transformação quando comparado à eficiência obtida com plasmídeos circulares. No entanto, a freqüência de transformantes multi-cópias aumentou significativamente. Juntos os procedimentos reportados aqui resultaram em processos mais eficientes de produção de protoplastos e transformação, onde o tempo necessário para o aparecimento dos transformantes e o número de inserções múltiplas foi reduzido.

Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum

Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold).

Fusões de protoplastos entre linhagens mutantes auxotróficas e morfológicas complementares de Penicillium griseoroseum e P. expansum foram induzidas por polietilenoglicol e íons cálcio (Ca2+). Fusionantes foram obtidos em meio mínimo e uma linhagem prototrófica, possivelmente diplóide, foi selecionada para a haploidização com o fungicida benomil. Diferentes linhagens recombinantes foram isoladas e caracterizadas quanto à presença de mutações auxotróficas e a produção de enzimas pectinolíticas. O fusionante prototrófico não apresentou maior atividade de pectinases em relação às linhagens parentais, entretanto, entre 29 recombinantes analisados, quatro apresentaram maiores atividades enzimáticas. O recombinante RGE27, o qual possui as mesmas mutações auxotróficas e morfológicas que a linhagem parental de P. griseoroseum, apresentou um aumento considerável na produção de poligalacturonase (3 vezes) e de pectina liase (1,2 vezes).

Morphological and molecular differentiation of the pectinase producing fungi Penicillium expansum and Penicillium griseoroseum

Two species from the genus Penicillium, Penicillium expansum and P. griseoroseum (Brasilian isolates) were characterized morphologic and molecularlly. Morphological variability was detected among isolates in regard to colony morphology and to conidia coloration. The molecular characterization was based on the RAPD markers, telomeric fingerprinting and ITS sequencing. A total of 78 RAPD primers were used and 8 presented differences in band patterns with 54 percent of the amplified polymorphic fragments. The monomorphic fragments of 600 bp (P. expansum) and 594 bp (P. griseoroseum) were amplified. The only internal transcribed spacer region variation detected between the two species was the additional six initial nucleotides. The analysis by telomeric fingerprinting showed polymorphism between both species and the chromosome minimal numbers estimated were three. The polymorphism observed in the organization of the subtelomeric region in the genome of two Penicillium species within the high homogeneous Penicillium subgenus is for the first time reported and perhaps can be employed in future phylogenetic studies.

Penicillium expansum e P. griseoroseum foram caracterizados morfológica e molecularmente. Variações na morfologia das colônias e coloração dos conídeos foram observadas entre os isolados. A caracterização molecular foi baseada em marcadores RAPD, sequenciamento da região do espaçador interno transcrito do DNA ribossomal e "fingerprinting" telomérico. Foi usado um total de 78 primers RAPD, sendo que 8 apresentaram 54 por cento de fragmentos de DNA polimórficos. Os produtos da amplificação da região ITS de P. expansum e P. griseoroseum foram de 600 e 594 pb, respectivamente. Não foi detectada nenhuma variação na seqüência de nucleotídeos dessa região, comparando-se P. expansum e P. griseoroseum, exceto em relação aos seis nucleotídeos iniciais adicionais. Observou-se a ocorrência de polimorfismo na organização da região subtelomérica no genoma destes fungos e estimou-se um número mínimo de três cromossomos para estas espécies. Este é o primeiro trabalho que descreve a existência de polimorfismo na organização da região subtelomérica do genoma de espécies de fungos pertencentes ao gênero Penicillium, altamente homogêneo, indicando uma possível utilização da abordagem empregada neste estudo para pesquisas filogenéticas futuras.

Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene

Penicillium brevicompactum é um fungo filamentoso que apresenta um potencial para a aplicacão industrial devido a sua eficiente producão de enzimas do complexo pectinolítico. Neste trabalho foi desenvolvido um sistema de transformacão heterólogo para P. brevicompactum baseado na complementacão de um mutante nitrato redutase. Mutantes nitrato redutase foram obtidos pela resistência ao clorato de sódio em uma taxa de 23,24per center. O mutante denominado 4457-18X foi escolhido para os experimentos de transformacão com o vetor pNH24, que contém o gene da nitrato redutase de Fusarium oxysporum. Uma freqüência de cerca de 3 transformantes/mg de DNA foi obtida utilizando-se o vetor pNH24 na forma circular e um aumento de cerca de 10 vezes nessa freqüência foi alcancado com a utilizacão desse vetor linearizado com a enzima de restricão Xba I. A análise dos transformantes pela técnica de hibridizacão revelou uma tendência do vetor linearizado diminuir o número de integracões em relacão ao vetor circular. A integracão foi aleatória e estável nos transformantes analisados. O estabelecimento de um sistema de transformacão para P. brevicompactum é essencial para a manipulacão genética desse microrganismo.

Pulsed field gel electrophoresis reveals chromosome length and number differences in Brazilian strains of Metarhizium Anisopliae

Cariótipos de oito linhagens selvagens do fungo entomopatogênico Metarhizium anisopliae var. anisopliae foram obtidos em gel, por eletroforese em campo pulsado. As linhagens foram isoladas de insetos provenientes de seis estados brasileiros. As moléculas de DNA cromossômico de três linhagens foram separadas em sete bandas e, de cinco linhagens, em oito bandas. Polimorfismo de tamanho cromossômico também foi observado. O tamanho do DNA cromossômico de todas as linhagens variou de 7,7 a 0,9 Mb, utilizando-se DNA cromossômico de Aspergillus nidulans como padrão. O tamanho do genoma total foi estimado em pelo menos 29,7 Mb. Algumas correlações entre semelhanças e diferenças no cariótipo eletroforético e a ocorrência do ciclo parassexual como também a especificidade com insetos hospedeiros foram discutidas.