Littérature scientifique sur le sujet « Bacillus amyloliquefaciens subsp »

The rhizosphere-isolated bacteria belonging to the Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus clades are an important group of strains that are used as plant growth promoters and antagonists of plant pathogens. These properties have made these strains the focus of commercial interest. Here, we present the draft genome sequence of B. methylotrophicus KACC 13105T ( = CBMB205T). Comparative genomic analysis showed only minor differences between this strain and the genome of the B. amyloliquefaciens subsp. plantarum type strain, with the genomes sharing approximately 95 % of the same genes. The results of morphological, physiological, chemotaxonomic and phylogenetic analyses indicate that the type strains of these two taxa are highly similar. In fact, our results show that the type strain of B. amyloliquefaciens subsp. plantarum FZB42T ( = DSM 23117T = BGSC 10A6T) does not cluster with other members of the B. amyloliquefaciens taxon. Instead, it clusters well within a clade of strains that are assigned to B. methylotrophicus, including the type strain of that species. Therefore, we propose that the subspecies B. amyloliquefaciens subsp. plantarum should be reclassified as a later heterotypic synonym of B. methylotrophicus.

The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42T (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42T to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7T but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42T, formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7T. DNA–DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7T and FZB42T yielded relatedness values of 63.7–71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7T and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42T ( = DSM 23117T = BGSC 10A6T), and B. amyloliquefaciens subsp. amyloliquefaciens subsp. nov., with the type strain DSM 7T( = ATCC 23350T = Fukumoto Strain FT), for plant-associated and non-plant-associated representatives, respecitvely. This is in agreement with results of DDH and M-CGH tests and the MALDI-TOF MS of cellular components, all of which suggested that the ecovars represent two different subspecies.

Selected bacterial isolates from the surface water of Buriganga river were characterized by morphological, biochemical characteristics and sequence-based PCR-amplified fragments of 16S rRNA. All isolates were rod shaped and Gram-positive. The isolates were confirmed as Chryseobacterium arthrosphaerae strain FDAARGOS 519, Bacillus cabrialesii strain TE 3, Bacillus tequilensis KCTC strain 13622(T), Bacillus amyloliquefaciens DSM 7 strain ATCC 23350, Bacillus subtilis strain E20 and Bacillus subtilis subsp. subtilis strain 168 based on sequence analysis. A phylogenetic tree was constructed that showed only one major cluster comprising of two sub-clusters grouping Chryseobacterium arthrosphaerae in one and Bacillus tequilensis in another. Chryseobacterium arthrosphaerae strain FDAARGOS 519 was susceptible to all antibiotics at different ranges, while Bacillus tequilensis KCTC strain 13622(T) and Bacillus amyloliquefaciens DSM 7 strain ATCC 23350 were found to be resistant to polymyxin only but sensitive to other antibiotics. However, Bacillus cabrialesii strain TE 3 was resistant to polymyxin and neomycin, while Bacillus subtilis subsp. subtilis strain 168 was resistant to polymyxin, vancomycin and rifampicin.

The Bacillus subtilis group encompasses the taxa Bacillus subtilis subsp. subtilis, B. licheniformis, B. amyloliquefaciens, B. atrophaeus, B. mojavensis, B. vallismortis, B. subtilis subsp. spizizenii, B. sonorensis, B. velezensis, B. axarquiensis and B. malacitensis. In this study, the taxonomic relatedness between the species B. axarquiensis, B. malacitensis and B. mojavensis was investigated. Sequence analysis of the 16S rRNA gene and the gene for DNA gyrase subunit B (gyrB) confirmed the very high similarities between these three type strains and a reference strain of B. mojavensis (>99 and >97 %, respectively). DNA–DNA hybridization experiments revealed high relatedness values between the type strains of B. axarquiensis, B. malacitensis and B. mojavensis and between these strains and a reference strain of B. mojavensis (83–98 %). Based on these molecular taxonomic data and the lack of phenotypic distinctive characteristics, Bacillus axarquiensis and Bacillus malacitensis should be reclassified as later heterotypic synonyms of Bacillus mojavensis.

There are two methods to identify the bacterial characteristic, namely biochemical analysis and the 16S ribosomal ribonucleic acid gene (16S rRNA) sequencing analysis. The research aimed to identify the cultured bacterial from ex-tin mining lakes by biochemistry analysis and molecular approach. Nine bacterial were cultured and isolated in nutrient agar and then biochemically characterized by microbact™ 12A and 24E (Oxoid) identification kits. In addition, molecular analysis by 16S rRNA gene was sequenced primer 1492R and primer 27F. Based on biochemistry analysis, these bacterial were identified as belonging to species of Bacillus amyloliquefaciens; Enterobacter gergoviae; Enterobacter aerogenes; Enterobacter agglomerans; and Nitrobacter spp. The sequence analysis in gene bank of NCBI indicated that these species had similarity with Klebsiella variicola strain F2R9 (Accession NR_025635.1); Enterobacter cloacae subsp. dissolvens strain LMG 2683 (Accession NR_044978.1); Serratia marcescens strain NBRC 102204 (Accession NR_114043.1); Bacillus marisflavi strain TF-11 (Accession NR_118437.1); Falsibacillus pallidus strain CW 7 (Accession NR_116287.1); Klebsiella pneumoniae strain DSM 30104 (Accession NR_117683.1); and Nitrobacter winogradskyi strain Nb-255 (Accession NR_074324.1). However, phylogenetic tree was constructed by Neighbor-Joining Test showed the cultured bacterial were not in the same clade and also with Salmonella enterica subsp. enterica strain LT2 (Accession NR_074910.1); Bacillus amyloliquefaciens strain BCRC 11601; and Escherichia coli strain NBRC 102203 (Accession NR_114042.1) as in group species and Micrococcus luteus strain NCTC 2665 (Accession NR_075062.2); Chloroflexus islandicus strain isl-2 (Accession NR_148571.2); Flavobacterium gondwanense (Accession M92278.1); and Cytophaga aurantiaca strain JM110 (Accession MN758870.1) as their out group.ABSTRAKTerdapat dua metode untuk mengidentifikasi karakteristik bakteri, yaitu analisis biokimia dan analisis sekuensing gen 16S ribosomal ribonucleic acid (16S rRNA). Karakterisasi bakteri telah dilakukan melalui analisis morfologi dan biokimia dan dikonfirmasimelalui pendekatan molekuler menggunakan sekuensing gen 16S ribosomal ribonucleic acid (16S rRNA). Penelitian ini bertujuan untuk mengidentifikasi bakteri yang dapat dikultur dari danau pascatambang timah melalui analisis biokimiawi dan pendekatan molekuler. Sembilan bakteria berhasil dikultur dan diisolasi di media nutrient agar dan kemudian secara biokimiawi dikarakterisasi menggunakan microbact™ 12A and 24E (Oxoid) identification kits. Lebih lanjut, analisis molekuler menggunakan gen 16S rRNA dilakukan sekuensing dengan primer 1492R dan primer 27F. berdasarkan analisis biokimia, bakteri-bakteri tersebut termasuk ke dalam spesies Bacillus amyloliquefaciens; Enterobacter gergoviae; Enterobacter aerogenes; Enterobacter agglomerans; dan Nitrobacter spp. Analisis blasting pada gene bank di NCBI mengindikasikan bahwa spesies-spesies tersebut memiliki kemiripan atau similaritas dengan Klebsiella variicola strain F2R9 (Accession NR_025635.1); Enterobacter cloacae subsp. dissolvens strain LMG 2683 (Accession NR_044978.1); Serratia marcescens strain NBRC 102204 (Accession NR_114043.1); Bacillus marisflavi strain TF-11 (Accession NR_118437.1); Falsibacillus pallidus strain CW 7 (Accession NR_116287.1); Klebsiella pneumoniae strain DSM 30104 (Accession NR_117683.1); dan Nitrobacter winogradskyi strain Nb-255 (Accession NR_074324.1). Namun, pohon filogenetik yang dikonstruksikan dengan Neighbor-Joining Test menunjukkan bahwa bakteri yang dikultur tersebut tidak berada pada clade dan juga dengan Salmonella enterica subsp. enterica strain LT2 (Accession NR_074910.1); Bacillus amyloliquefaciens strain BCRC 11601; dan Escherichia coli strain NBRC 102203 (Accession NR_114042.1) yang digunakan sebagai spesies in group species maupun Micrococcus luteus strain NCTC 2665 (Accession NR_075062.2); Chloroflexus islandicus strain isl-2 (Accession NR_148571.2); Flavobacterium gondwanense (Accession M92278.1); dan Cytophaga aurantiaca strain JM110 (Accession MN758870.1) sebagai out groupnya.

Orientador : Prof. Dr. Átila Francisco Mógor
Coorientador : Profª. Drª. Lucimeris Ruaro
Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Agronomia. Defesa: Curitiba, 14/06/2016
Inclui referências: f 24-28;31-32;42-45;53-55;68-71;84-88;93-95
Área de concentração: Produção vegetal
Resumo: O tomateiro é uma cultura amplamente difundida, seus frutos podem ser utilizados in natura ou industrializados. Portanto possui relevância econômica e importância social pois seus derivados passam por uma extensa cadeia produtiva que gera inúmeros empregos. Atualmente com uma maior conscientização ambiental e também visando a saúde, o uso de tecnologias biológicas que incrementem a produtividade sem aditivos químicos, são alternativas desejáveis. Neste contexto o uso de bactérias promotoras de crescimento vegetal vem de encontro a estas necessidades. A bactéria Bacillus amyloliquefaciens subsp. plantarum FZB42, que já tem caracterizada algumas habilidades de interesse agronômico, foi o alvo utilizado no estudo da interação planta x bactéria. Sendo assim, os objetivos deste estudo foram: (i) investigar na bactéria características relacionadas à promoção do crescimento vegetal; (ii) verificar alterações morfométricas das plantas de tomateiro inoculadas com FZB42 na germinação de sementes, na produção de mudas e no crescimento das plantas até 60 dias em sistema orgânico em diferentes doses (1,5x109; 6,0x1010; 2,4x1011 bactérias mL-1); (iii) analisar alterações bioquímicas e nutricionais em plantas de tomateiro; (iv) avaliar a produção de tomates mediante inoculação da bactéria nas doses (1,5x109; 6,0x1010). FZB42 apresentou resultados positivos para produção de sideróforos e compostos indólicos com e sem suplementação de triptofano. Sementes inoculadas não tiverem seu percentual de germinação alterado. Na produção de mudas, a dose de 6,0x1010, foi a mais estimuladora do crescimento vegetal, com incrementos na parte aérea das cultivares Cereja 261, Santa Clara I-5300, Santa Cruz Kada Gigante e Serato F1, bem como aumentou os teores de clorofila. As mudas das duas últimas cultivares, na mesma dose, também tiveram o sistema radicular estimulado e os teores de açúcares solúveis e proteínas solúveis aumentados. A dose de 1,5x109 reduziu o crescimento das mudas. Plantas com 60 dias após semeadura, de 'Cereja 261', 'Santa Clara I-5300' e 'Serato F1' apresentaram-se com maior parte aérea e raízes quando inoculadas com 6,0x1010. Plantas de 60 dias submetidas a 1,5x109 tiveram estímulos mais discretos e quando inoculadas com 2,4x1011 os aspectos biométricos foram diminuídos. Nesta etapa o perfil metabólico dos cultivares não seguiu um padrão, pois estavam sujeitos às variação morfométricas devido às diferenças de estadio fenológico. O cultivar Serato F1 foi conduzido à campo em cultivo protegido. Aos 135 dias após plantio (em plena frutificação) apresentou, nas folhas, açúcares solúveis totais e aminoácidos livres totais em maiores quantidades nas plantas inoculadas com as doses de 1,5x109 e 6,0x1010. Já os frutos maduros das plantas inoculadas, também colhidos no mesmo momento, apresentaram mais proteínas solúveis totais e aminoácidos livres totais. Neste mesmo período a análise de macro e micro nutrientes revelou que plantas inoculadas tinham maiores teores de nitrogênio, ferro e manganês. A dose de 6,0x1010 diminuiu os níveis de cobre e aumentou zinco. Quanto aos aspectos produtivos, todos os tratamentos com plantas inoculadas apresentaram cerca de quarto frutos a mais por planta, sendo estes frutos de maior calibre e mais pesados que o controle não inoculado. Isso refletiu em uma produção de cerca de 1 kg a mais por planta, o que considerando 12,820 plantas ha-1, representa um incremento de 11,76 e 13,23 ton ha-1 nas doses de 1,5x109 e 6,0x1010, respectivamente. Portanto, FZB42 na dose de 6,0x1010 foi mais adequado para estímulo ao crescimento vegetal na fase de produção de mudas. Em termos de produtividade ambas as doses 1,5x109 e 6,0x1010 foram eficazes. Sendo assim, FZB42 na dose de 6,0x1010 bactérias mL-1 foi efetivo na promoção de crescimento durante todo o ciclo do tomateiro e com isso demonstrou potencial como inoculante ou bio-fertilizantes proporcionando alterações metabólicas e nutricionais. Palavras-chave: Bactérias promotoras de crescimento vegetal. Inoculante. Bio-fertilizante. Produtividade. Bacillus amyloliquefaciens. Solanum lycopersicum.
Abstract: The tomato is a widespread crop, its fruits can be used as a raw material for processed food. Therefore it has economic relevance and social importance because is the basis of an extensive production chain that generates numerous jobs. Currently greater environmental awareness and also aimed at health, the use of biological technologies that increase productivity without chemical additives, are desirable alternatives. In this context the use of plant growth promoting bacteria comes to attend these needs. The bacteria Bacillus amyloliquefaciens subsp. plantarum FZB42, which already has characterized some desirable agronomically desirable effects, was the aim at this study of plant x bacteria interaction. Thus, the objectives of this study were: (i) investigate the bacteria characteristics related to the promotion of plant growth; (ii) verify morphometric changes of tomato plants inoculated with FZB42 on seed germination, in the production of seedlings, and plant growth to 60 days after sowing in organic system at different doses (1.5x109; 6.0x1010; 2.4x1011 bacteria mL-1); (iii) analyze biochemical and nutritional changes in tomato plants; (iv) evaluate the production of tomatoes by inoculation of bacteria in doses (1.5x109; 1,6x105). FZB42 tested was positive for siderophore production and indolec compounds with and without tryptophan supplementation. Inoculated seeds have not changed their germination percentage. In the production of seedlings, the dose of 1.6x105 was the most stimulating plant growth, with increases in the shoots of 'Cherry 261', 'Santa Clara I-5300', 'Santa Cruz Kada Gigante' and 'Serato F1', as well as increased levels of chlorophyll. The last two cultivars seedlings at the same dose also had their roots stimulated and showed increaes in total soluble sugars and total soluble proteins. The dose of 2.4x1011 reduced seedling growth. Plants 60 days after sowing, the 'Cherry 261,' 'Santa Clara I-5300' and 'Serato F1' were improved their shoots and roots growth when inoculated with 6.0x1010. Plants with 60 days inoculates with 1.5x109 had more discrete stimuli and when inoculated with 2.4x1011 the growth were diminished. At this stage the metabolic profile of cultivars did not follow a pattern because they were subject to the morphometric variation due differences in phenological stages. The cultivar Serato F1 was cultivated in greenhouse. At 135 days after planting (in full fruiting) the leaves presented total soluble sugars and total free amino acids in a larger amount in the plants inoculated with doses of 1.5x109 and 6.0x1010. In addition, the ripe fruits of inoculated plants, harvested at this time, had more total soluble protein and total free amino acids. In the same period, the analysis of macro and micronutrients on leaves showed that inoculated plants had higher levels of nitrogen, iron and manganese. The dose of 6.0x1010 decreased copper and increased zinc levels. All inoculated plants showed the increment of around four fruits per plant, which fruits with greater caliber and heavier than uninoculated control. This reflected in an increasing of about 1 kg at more per plant, considering that 12,820 plants ha-1, representing an increase of 11.76 tons ha-1 and 13.23 tons ha-1 at the doses of 1.5x109 and 6.0x1010 respectively. Therefore, Bacillus amyloliquefaciens FZB42 at 6.0x1010 dose was more efficient for stimulating plant growth in seedling production. In terms of yield, both 1.5x109 and 6.0x1010, doses were effective. Thus, FZB42 at a dose of 6.0x1010 bacteria mL-1 was effective in promoting growth throughout the tomato cycle and it showed potential as inoculant or bio-fertilizer providing nutritional and metabolic changes. Key-words: Plant growth promoting bacteria. Inoculation. Biofertilizers. Productivity. Bacillus amyloliquefaciens. Solanum lycopersicum.

Understanding how soil-borne microorganisms can modulate the plant defence responses and which factors affect rhizosphere multitrophic interactions is crucial to improve the efficacy of biopesticides in agriculture. From this perspective, whole-genome sequencing is a powerful tool to characterize the bacterial strains of agronomic interest. Among these, Bacillus amyloliquefaciens subsp. plantarum strain S499 is a plant-beneficial rhizobacterium that shows direct antagonistic properties against phytopathogenic fungi and, in addition, a remarkable efficacy as elicitor of induced systemic resistance (ISR) in plants. In these activities, the production of bioactive secondary metabolites, such as cyclic lipopeptides belonging to the fengycin, iturin and surfactin families, is involved. By sequencing, assembling and annotating S499 genome, we identified the principal genes involved in root colonization, plant-growth promotion and biocontrol activities. These genes share a high percentage of nucleotide identity with their homologs in the strain FZB42, the type strain of the bacterial subspecies. One of the main genetic elements distinguishing S499 from FZB42 is the presence of extrachromosomal DNA (plasmid pS499). This small rolling circle plasmid was unknown before S499 genome sequencing, which also allowed to identify on pS499 the genes encoding a Rap-Phr regulatory system involved in quorum sensing. Through a plasmid-curing approach, we carried out a functional characterization of pS499. First, we studied the impact of the plasmid loss on the bacterial physiology, by comparing the behaviours of S499, its plasmid-cured derivative, S499 P-, and FZB42 on Luria-Bertani (LB) medium. Growth rate, extracellular proteolytic activity and the regulation of lipopeptide production were significantly affected in S499 P-. In agreement with an increased release of surfactins, swarming motility improved after curing, whereas biofilm production was reduced in vitro. When the evolution of bacterial populations was compared in planta, pS499 seemed not to influence the root colonization ability, although we observed an over-production of surfactins by S499 P- also on tomato roots. The quantification of the relative expression of srfA and rap genes suggested an inhibitory effect of the plasmid-encoded Rap-Phr system on surfactin synthesis. Moreover, on LB, the antagonistic effect against phytopathogenic fungi was limited for S499 P-, most probably due to a verified reduction of iturin secretion. Although less clearly, an impact of plasmid curing on the biocontrol ability was observed also on a medium (RE) that reproduced the typical composition of plant root exudates. Globally, our results show that pS499 differently modulates S499 phenotype depending on the nutritional context. More evidences are required to prove that pS499 is relevant for the fitness of the rhizobacterium in its natural environment.

Understanding how soil-borne microorganisms can modulate the plant defence responses and which factors affect rhizosphere multitrophic interactions is crucial to improve the efficacy of biopesticides in agriculture. From this perspective, whole-genome sequencing is a powerful tool to characterize the bacterial strains of agronomic interest. Among these, Bacillus amyloliquefaciens subsp. plantarum strain S499 is a plant-beneficial rhizobacterium that shows direct antagonistic properties against phytopathogenic fungi and, in addition, a remarkable efficacy as elicitor of induced systemic resistance (ISR) in plants. In these activities, the production of bioactive secondary metabolites, such as cyclic lipopeptides belonging to the fengycin, iturin and surfactin families, is involved. By sequencing, assembling and annotating S499 genome, we identified the principal genes involved in root colonization, plant-growth promotion and biocontrol activities. These genes share a high percentage of nucleotide identity with their homologs in the strain FZB42, the type strain of the bacterial subspecies. One of the main genetic elements distinguishing S499 from FZB42 is the presence of extrachromosomal DNA (plasmid pS499). This small rolling circle plasmid was unknown before S499 genome sequencing, which also allowed to identify on pS499 the genes encoding a Rap-Phr regulatory system involved in quorum sensing. Through a plasmid-curing approach, we carried out a functional characterization of pS499. First, we studied the impact of the plasmid loss on the bacterial physiology, by comparing the behaviours of S499, its plasmid-cured derivative, S499 P-, and FZB42 on Luria-Bertani (LB) medium. Growth rate, extracellular proteolytic activity and the regulation of lipopeptide production were significantly affected in S499 P-. In agreement with an increased release of surfactins, swarming motility improved after curing, whereas biofilm production was reduced in vitro. When the evolution of bacterial populations was compared in planta, pS499 seemed not to influence the root colonization ability, although we observed an over-production of surfactins by S499 Palso on tomato roots. The quantification of the relative expression of srfA and rap genes suggested an inhibitory effect of the plasmidencoded Rap-Phr system on surfactin synthesis. Moreover, on LB, the antagonistic effect against phytopathogenic fungi was limited for S499 P-, most probably due to a verified reduction of iturin secretion. Although less clearly, an impact of plasmid curing on the biocontrol ability was observed also on a medium (RE) that reproduced the typical composition of plant root exudates. Globally, our results show that pS499 differently modulates S499 phenotype depending on the nutritional context. More evidences are required to prove that pS499 is relevant for the fitness of the rhizobacterium in its natural environment.

We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...