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Genetic transformation is a valuable and essential method that provides powerful insights into the gene function of microorganisms and contributes to the construction of engineered bacteria. Here, we developed a novel genetic transformation system to easily knock out a highly GC‐rich gene (74.71% GC) from Burkholderia pyrrocinia JK‐SH007, a biocontrol strain of poplar canker disease. This system revealed a reliable selectable marker (trimethoprim resistance gene, Tmp) and a simplified, efficient transformation method (6,363.64 CFU/μg, pHKT2) that was developed via freeze‐thawing. The knockout recombineering of B. pyrrocinia JK‐SH007 was achieved through a suicide plasmid with a three‐fragment mutagenesis construct. The three‐fragment cassette for mutagenesis was generated by overlap extension and touchdown PCRs and composed of Tmp flanked by GC‐rich upstream and downstream fragments from B. pyrrocinia JK‐SH007. The mutant strain (ΔBpEG), which was verified by PCR, lost 93.3% of its ability to degrade carboxymethyl cellulose over 40 days. Overall, this system may contribute to future research on B. pyrrocinia traits., A novel genetic transformation system was developed to easily knock out a highly GC‐rich gene (74.7% GC) from Burkholderia pyrrocinia JK‐SH007 by homologous recombination with freeze‐thawing transformation.

With the development of marine biorefinery concept, utilisation of algal waste during industrial processing as well as some “green tide” waste biomass has become an important research topic. In this work, a single-step microwave process was used to hydrolyse Laminaria japonica processing waste (LJW) and Enteromorpha prolifera (EP), producing a growth medium suitable for microbial cultivation. The medium contained a range of mono- and polysaccharides as well as macro- and micronutrients that could be used by the microbes. The cultivation behavior of three plant growth-promoting rhizobacteria (PGPR) strains (Bacillus subtilis strain Tpb55, Bacillus amyloliquefaciens strain Cas02, and Burkholderia pyrrocinia strain Lyc2) in the two media were investigated. LJW hydrolysate from 180 °C and EP hydrolysate from 150 °C performed better cultivation efficiency than those hydrolysates from other microwave conditions. Saccharide analysis showed that microbes metabolized some monosaccharide such as glucose, mannose during cultivation, leaving polysaccharide unused in the medium. Furthermore, hydrolysate-strain cultivation mixtures were applied to pepper growth. The EP hydrolysate-Cas02 broth showed better plant growth-promoting effect compared to other treatments, which might be attributed to the higher indole-3-acetic acid (IAA) production of Cas02 in the EP hydrolysate. This work shed lights on the conversion of algal waste to PGPR biomass as well as the co-application of algal hydrolysates- strains cultivation broth for a better plant growth promotion.

Multifunctional microorganisms can contribute to the development of plant root systems and positively affect the grain yield of crops. However, studies of the use of these microorganisms to benefit root development in rice plants are scarce. The objective of this study was to determine the effect of rhizobacterial inoculation of the seeds on the root development of upland rice seedlings. The experiment was carried out in the agricultural microbiology laboratory of Embrapa Rice and Beans, and we adopted a completely randomized design with eight replications. The treatments consisted of the inoculation of rice seeds with the rhizobacteria 1. Pseudomonas fluorescens (BRM 32111), 2. Burkholderia pyrrocinia (BRM 32113), 3. Serratia sp. (BRM 32114), 4. Bacillus sp. (1301), 5. Azospirillum brasilense (Ab-V5), 6. Azospirillum sp. (1381), and a 7. control treatment (without microorganisms). Treatment using the microorganism Azospirillum sp. (1381) provided the longest roots, greatest root volume and highest total dry mass of roots, at 86.34, 111.53 and 29.41%, respectively, significantly superior to the control treatment. In addition, treatments Ab-V5 and Bacillus sp. (1301) resulted in a root diameter of 13.81 and 13.06%, respectively, larger than that of the control treatment. The use of these multifunctional microorganisms was efficient for promoting the root development of rice seedlings. Made available in DSpace on 2021-11-30T15:00:57Z (GMT). No. of bitstreams: 1 sca-2021-2.pdf: 1033152 bytes, checksum: 7898838db384cedd1154d3ae03347118 (MD5) Previous issue date: 2021

Volatile organic compounds (VOCs) play important roles in the regulation of plant growth and pathogen resistance. However, little is known about the influence of VOCs released from endophytic strains (Burkholderia pyrrocinia strain JK-SH007) on controlling pathogens or inducing systemic resistance in poplar. In this study, we found that VOCs produced by strain JK-SH007 inhibit three poplar canker pathogens (Cytospora chrysosperma, Phomopsis macrospora, and Fusicoccum aesculi) and promote defense enzyme activity and malondialdehyde (MDA) and total phenol (TP) accumulation. Thirteen kinds of VOC components were identified using the solid-phase microextraction combined with gas chromatography-mass spectrometry method. Dimethyl disulfide (DMDS) accounted for the largest proportion of these VOCs. Treatments of poplar seedlings with different volumes of VOC standards (DMDS, benzothiazole, dimethylthiomethane, and phenylacetone) showed that DMDS had the greatest effects on various defense enzyme activities and MDA and TP accumulation. We also found that the inhibitory effect of the VOCs on the three pathogens was gradually enhanced with increasing standard volume. Moreover, the treatment of samples with DMDS significantly reduced the severity and development of the disease caused by three poplar canker pathogens. Comparative transcriptomics analysis of poplar seedlings treated with DMDS showed that there were 1,586 differentially expressed genes in the leaves and stems, and quantitative PCR showed that the gene expression trends were highly consistent with the transcriptome sequencing results. The most significant transcriptomic changes induced by VOCs were related to hormone signal transduction, transcriptional regulation of plant–pathogen interactions, and energy metabolism. Moreover, 137 transcription factors, including members of the ethylene response factor, NAC, WRKY, G2-like, and basic helix-loop-helix protein families, were identified to be involved in the VOC-induced process. This study elucidates the resistance induced by Burkholderia pyrrocinia strain JK-SH007 to poplar canker at the molecular level and can make possible a new method for the comprehensive prevention and control of poplar disease.

Zinc (Zn) contamination in soil has been a serious problem world widely. Plant growth-promoting bacterium (PGPB) could have potential applications in Zn bioremediation. In this study, Burkholderia ...

This report examines the soluble expression of a novel feruloyl esterase, BpFae, from Burkholderia pyrrocinia in Escherichia coli overexpression systems using three expression vectors, pET28a, pCold-TF and pGEX-4T-1. BpFae was overexpressed as insoluble inclusion bodies when pET28a was used as the expression vector. BpFae was overexpressed in the soluble form when using pCold-TF; however, the overexpressed protein showed negligible activity. Recombinant BpFae was produced in the soluble form and active when E. coli cells were transformed with the pGEX-4T-1-BpFae vector. Optimal conditions for soluble overexpression of recombinant BpFae were determined, and the highest activity of recombinant BpFae was 2.54 U mL−1. Multiple sequence alignment, phylogenetic analysis and construction of a three-dimensional model indicated that BpFae is a unique FAE from B. pyrrocinia with underlying research value.

Increasing the expression level of purple acid phosphatases (PAPs), which hydrolyze organic phosphate to inorganic phosphate (Pi), is an important response to Pi starvation in plants. PAPs are widely distributed in eukaryotes like mammals and plants but distributed in limited microorganisms. A PAP-like protein named Pap9 from Burkholderia pyrrocinia CH-67 was isolated. The enzyme had optimal activity at atypical pH of 8.5 and temperature of 85°C. When pap9 was overexpressed in Arabidopsis thaliana, the APase activity in the transgenic plants was about 15% to 40% higher under acidic conditions and had an approximately four-fold increase under alkaline conditions compared with wild type. Overexpression of pap9 in plants enhanced APase activity under Pi-deficient conditions more than under Pi-sufficient conditions. In-gel assays revealed that Pap9 exists as a monomer in transgenic plants. Root surface-associated APase activity in transgenic plants increased dramatically during acidic and Pi-deficient conditions. These results indicated that bacterial Pap9 in transgenic plants was secreted onto the root surface and released into the rhizosphere. The transgenic plants have significantly enhanced biomass that have the potential for use in biotechnological applications.

Burkholderia pyrrocinia CAB08106-4은 Sclereotium cepivorum와 Sclereotium sp.균주에 의해 발생하는 마늘 흑색썩음균핵병에 대한 항박테리아 효능을 가지고 있다. 이는 Sclereotium cepivorum와 Sclereotium sp.에 의해 발생하는 마늘 흑색썩음균핵병을 포함한 다앙한 식물병원체를 관리하고 예방하는데 있어 친환경적인 미생물 제품이다. 이번 시험의 목적은 Burkholderia pyrrocinia CAB08106-4를 피부, 안점막 투여시에 나타나는 국소자극성과 피부감 작성에 대해 평가하고자 한다. 피부, 안점막 자극성 시험에서 NewZealand White계 토끼의 피부와 안점막에 투여를 하고, 피부자극성시험은 72시간 동안, 안점막자극성시험은 7일 동안 매일 임상증상 관찰을 하였으며, 시험물질 투여 전, 투여 후 3 일 및 안점막자극성시험은 7 일째에 체중을 측정하여 체중증가량을 평가하였다. 그 결과, 전체 시험기간 동안 일반증상 및 피부자극성, 안점막자극성이 관찰되지 않았고, 체중도 정상적으로 증가한 것으로 관찰되었다. 피부감작성시험에서 Hartley계 기니피그에 투여를 하고, 시험물질 야기 후 24시간과 48시간에 투여부위의 피부를 관찰하였다. 그 결과, 시험물질 투여에 의한 일반증상이 관찰되지 않았고, 정상적인 체중 증가가 관찰되었으며, 홍반 및 부종 등의 피부반응이 관찰되지 않아 감작지수와 빈도지수가 각각 0과 0%로 산출되어 I등급으로 평가되었다. 이상의 결과를 통해 피부자극성시험, 안점막자극성시험 및 피부감작성시험에서 시험물질은 피부자극성과 안점막자극성을 일으키지 않았고, 피부감작성도 일으키지 않아 문제가 없는 것으로 평가되었다.

The Burkholderia pyrrocinia Lyc2 strain isolated from healthy plant rhizosphere showed significant antimicrobial activities against a variety of plant pathogens. In this study, a random mutation library was constructed using an EZ-Tn5 transposome kit and Erwinia amylovora was used as an indicator to screen for mutants with defective antibacterial activity. The transposon gene was verified in the chromosome of the Lyc2 strain using polymerase chain reaction (PCR). The gene that was disrupted by transposon was amplified by rescue cloning for functional and bioinformatics analyses. Antibacterial analysis indicated that the mutant Lyc2-MT2918 was defective in antibacterial activity. Sequence alignment of the mutant suggested that the disrupted gene Glu-2918 was homologous to the glutathione (GSH) synthase gene Bamb-2918 of strain B. ambifaria AMMD. Genetic functional analysis and complementary assay of the disrupted gene, which was predicted to encode GSH synthase, indicated the essential role of the Glu-2918 gene in the antibacterial activity of strain Lyc2.

Leaf blast is the main rice disease in the world causing significant losses in productivity. Blast integrate management (BIM) requires the use of genetic resistance, cultural practices, and chemical control, although for sustainable BIM, the insertion of biological agents may be the fourth component for. The objective of this work was to test three formulations of Burkholderia pyrrocinia (BRM32113) previously selected and to verify the effectiveness in resistance induction and blast control in rice. Two experiments were carried out, in a completely randomized design with three replications, in the greenhouse (E1 and E2). E1 aimed to select the best treatment for suppressing leaf blast severity and activating plant defense mechanisms. It was composed of 8 treatments: (1) formulated 11+ B. pyrrocina × Magnaporthe oryzae; (2) formulated 17+ B. pyrrocina × M. oryzae; (3) formulated 32+ B. pyrrocina × M. oryzae; (4) formulated 11 × M. oryzae; (5) B. pyrrocinia 17 × M. oryzae; (6) formulated 32 × M. oryzae; (7) B. pyrrocina × M. oryzae; (8) M. oryzae; (9) control (water). E2 aimed to investigate the effect of the best treatments, for the promotion of plant growth and suppression of leaf blast by calculating AUDPC. It was composed of 6 treatments: (1) formulated 11+ B. pyrrocina × M. oryzae; (2) formulated 32+ B. pyrrocina × M. oryzae; (3) formulated 11 × M. oryzae; (4) formulated 32 × M. oryzae; (5) B. pyrrocina × M. oryzae; (6) water. And after, we did two assays aimed to localize this biological agent after application at seed, soil, and rice plant. In E1, formulated 11+ B. pyrrocinia and 32+ formulated and B. pyrrocina were the best, suppressing leaf blast by up to 97% and providing the significant increase of the enzymes β-1,3-glucanase, chitinase, phenylalanine ammonia lyase, lipoxygenase, and salicylic acid at 24 h and 48 h after inoculation with M. oryzae. In E2, treatments formulated 11+ B. pyrrocinia, formulated 32+ B. pyrrocinia, and B. pyrrocina provided more significant increases in growth promotion and reduced area under disease progress curve. B. pyrrocinia was detected in the rice plant for 18 days, predominantly in the root system (internal and external). The use of B. pyrrocinia formulations based on sugarcane molasses and glycerol can be an essential strategy for sustainable management. Although all the benefits come from these sustainable formulations, the adoption by commercial biological segment depends on an established formulation process. It seems that all the results showed here by this research will be readily assimilated by startups of the organic segment.

Para o crescimento e desenvolvimento as plantas, na natureza, contam com os benefícios de microrganismos residentes em sua rizosfera. Rizobactérias promotoras de crescimento vegetal (RPCV) são exemplos desses microrganismos que interagem de forma benéfica com espécies vegetais. Portanto, a inoculação de sementes com RPCV pode representar uma alternativa importante e sustentável para melhorar o desenvolvimento inicial e a produção de plantas cultivadas. Objetivou-se com esse estudo determinar o efeito da microbiolização de sementes com RPCV no comprimento radicular de plântulas de arroz irrigado. Em condições controladas, conduziram-se dois experimentos com cultivares de arroz irrigado (BRS Catiana e A 702 CL), em delineamento inteiramente casualizado, com sete tratamentos e 10 repetições. Os tratamentos constituíram-se de seis microrganismos: BRM32109 (Bacillus sp.), BRM32110 (Bacillus sp.), BRM32111 (Pseudomonas sp.), BRM32112 (Pseudomonas fluorescens), BRM32113 (Burkholderia pyrrocinia), BRM32114 (Serratia sp.) e um controle (sem microrganismo). Os dados foram submetidos à análise de variância, as médias entre os tratamentos com RPCV foram comparadas pelo teste LSD (p≤0,05) e, os tratamentos com microrganismos foram comparados individualmente com o controle pelo teste de Dunnett (p≤0,05). Somente a cultivar BRS Catiana apresentou diferença entre os tratamentos. Os maiores comprimentos radiculares foram apresentados pelas sementes tratadas com os isolados BRM32110, BRM32112, BRM32109 e BRM32114. Para ambas cultivares houve significância quando comparado microrganismos e controle. Destaque para as sementes da cultivar BRS Catiana, que quando tratadas com os isolados BRM32110 e BRM32112 apresentaram aumento médio de 25,7% no comprimento radicular, em comparação com o tratamento controle. Para a cultivar A 702 CL, todos os microrganismos proporcionaram incrementos com um valor médio de 31% no comprimento radicular das plântulas de arroz. Com base nos resultados pode-se inferir que as RPCV proporcionam incrementos significativos no comprimento radicular de plântulas de cultivares de arroz irrigado.

Allelopathic compounds reduce the growth and productivity of upland rice plants, especially in consecutive plantations. The rhizobacteria Pseudomonas fluorescens BRM-32111 and Burkholderia pyrrocinia BRM-32113 have been recorded as growth promoters in rice. This study was developed to understand the effect of the application of rhizobacteria on upland rice plants in consecutive plantations. Experiments were conducted in a completely randomized design with four replications of four treatments: rice seed inoculated with P. fluorescens BRM-32111, rice seed inoculated with B. pyrrocinia BRM-32113 (both sown on soil with rice residue), non-inoculated plants sown on soil with rice residue (control with residue (WR)), and non-inoculated plants on soil with no residue (NR). Roots and seedling growth were adversely affected by allelopathic compounds in control WR plants. Plants inoculated with rhizobacteria P. fluorescens BRM-32111 or B. pyrrocinia BRM-32113 induced an increase of 88% in biomass, 3% in the leaf area, 40% in length, 67% in root biomass, 21% in chlorophyll a, 53% in chlorophyll (a+b), 50% in rate of carbon assimilation (A), 227% in A/rubisco carboxylation efficiency (Ci) and 63% in water use efficiency (WUE) compared to control WR plants. These results indicate that rhizobacteria P. fluorescens BRM-32111 and B. pyrrocinia BRM-32113 increase the tolerance of rice plants to stress from allelochemicals. There are possible practical agricultural applications of these results for mitigating the effects of environmental allelochemistry on upland rice.

Rice the main source of energy to maintain the metabolism for almost four billion people worldwide. Riceplants treated with multifunctional microorganisms may be a good wayfor sustainable improvement of crop grain yield. Aimedstudy the effect of types and ways of microorganisms application in tropical lowland rice production, evaluated by biomass production and gas exchange. The greenhouse trial was in a completely randomized design, in factorial scheme 7 x 3 + 1, with four replication. Treatments were the combination of seven microorganisms: Bacillussp. (BRM32109 e BRM32110); Pseudomonas fluorescens(BRM32111); Pseudomonassp. (BRM32112); Burkholderia pyrrocinia(BRM32113); Serratiasp. (BRM32114) and Trichoderma asperellumpool (UFRA.T06 + UFRA.T09 + UFRA.T12 + UFRA.T52), with three forms of application (1. microbiolized seed; 2. microbiolized seed + soil drenched with microorganism at eight and 15 days after sowing (DAS)and 3. microbiolized seed + microorganism spray plant at eight and 15 DAS). Microbiolized seed was the best form of application,which allowed highest instantaneous carboxylation efficiency and yield of biomassproduction in lowland rice. Rhizobcteria BRM32114, followed by BRM32111, BRM32112 and fungi T. asperellumimproved, an average, 17% in instantaneous carboxylation efficiency and 20% in dry shoot biomass production of lowland rice compared to the control plants. Made available in DSpace on 2021-11-30T13:00:35Z (GMT). No. of bitstreams: 1 ca-2021.pdf: 499279 bytes, checksum: 0655c967052fac01e7750c100da28826 (MD5) Previous issue date: 2021

Plant growth-promoting rhizobacteria that produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can promote plant growth and enhance abiotic stress tolerance. In this study, Burkholderia pyrrocinia strain P10, with an ACC deaminase activity of 33.01-µmol/h/mg protein, was isolated from the tea rhizosphere and identified based on morphological, biochemical, and molecular characteristics. In addition to its ACC deaminase activity at pH 5.0–9.0 and in response to 5% NaCl and 20% polyethylene glycol, strain P10 can also solubilize phosphorus compounds, produce indole-3-acetic acid, and secrete siderophores. Pot experiments revealed that strain P10 can significantly enhance peanut seedling growth under saline conditions (100- and 170-mmol/L NaCl). Specifically, it increased the fresh weight and root length of plants by 90.12% and 79.22%, respectively, compared with high-salt stress. These results provide new insights into the biological characteristics of Burkholderia pyrrocinia, which may be useful as a bio-fertilizer.

Burkholderia pyrrocinia B1213, a novel microbe isolated from a Baijiu-producing environment, displayed strong cellulolytic activity on agar plates with glucan as the carbon source and had an activity of 674.5 U/mL after culturing with barley. Genome annotation of B. pyrrocinia identificated a single endoglucanase (EG)-encoding gene, designated as BpEG01790. The endoglucanase BpEG01790 shows 98.28% sequence similarity with an endo-β-1,4-glucanase (EC 3.2.1.4) from Burkholderia stabilis belonging to glycoside hydrolase family 8 (GH8). The gene BpEG01790 has an open reading frame of 1218 bp encoding a 406 amino acid (AA) residue protein (43.0 kDa) with a 40-AA signal peptide. BpEG01790 was successfully cloned into pET28a( +) with and without the signal peptide; however, attempts to overexpress this protein in Escherichia coli BL21(DE3) cells using this expression system failed. BpEG01790 was also cloned into the pCold TF vector. Active BpEG01790 was successfully overexpressed with or without the signal peptide using the pCold TF vector expression system and E. coli BL21 (DE3) cells. Overexpression of recombinant BpEG01790 without the signal peptide was higher compared with the construct that included the signal peptide. Optimization of culture conditions improved the enzyme activity by 12.5-fold. This is the first report describing the heterologous soluble overexpression of an EG belonging to GH8 from B. pyrrocinia using TF as a molecular chaperone.

Multifunctional microorganisms can contribute significantly to improve plant performance and are innovative technology for sustainable development. Thus, objective of this study was to determine the effect of bioagents applied alone or in mix on the performance of upland rice. The experiment was conducted in a greenhouse in a completely randomized design with four replications. The 26 treatments consisted of the rhizobacteria Bacillus sp. (BRM 32109, BRM 32110 and 1301), Azospirillum sp. (1381), Azospirillum brasilense (Ab-V5), Pseudomonas sp. (BRM 32112), Pseudomonas fluorescens (BRM 32111), Burkholderia pyrrocinia (BRM 32113), Serratia sp. (BRM 32114), and a fungal genus formed by a pool of Trichoderma asperellum (T-06, T-09, T-12, and T-52), applied alone or in mix, plus a control treatment without microorganism application. The most effective treatments were the mixes of microorganisms 1301 + Ab-V5 and BRM 32114 + pool of Trichoderma asperellum, as they provided an average increase of 123 and 88% in the number of panicles and 206 and 167% in the grain yield of upland rice plants, respectively. Mixes of 1301 + Ab-V5, BRM 32114 + Trichoderma asperellum pool, BRM 32110 + BRM 32114, BRM 32110 + Ab-V5, 1301 + BRM 32110 and 1381 + Trichoderma asperellum pool also provided better morphophysiological performance in rice plants (photosynthetic rate, carboxylation efficiency, number of tillers, shoot dry biomass and nutrient content in shoot and root). Therefore, the use of multifunctional microorganisms in the management of upland rice was efficient in its ability to provide better development of plants. RESUMO Microrganismos multifuncionais podem contribuir significativamente para melhorar o desempenho das plantas, sendo tecnologia inovadora quando se visa o desenvolvimento sustentável. Assim, objetivou-se determinar o efeito de microrganismos multifuncionais aplicados isolados ou em mix sobre o desempenho do arroz de terras altas. O experimento foi conduzido em casa de vegetação, em delineamento inteiramente casualizado, com quatro repetições. Os 26 tratamentos consistiram das rizobactérias Bacillus sp. (BRM 32109, BRM 32110 e 1301), Azospirillum sp. (1381), Azospirillum brasilense (Ab-V5), Pseudomonas sp. (BRM 32112), Pseudomonas fluorescens (BRM 32111), Burkholderia pyrrocinia (BRM 32113), Serratia sp. (BRM 32114), e um gênero fúngico formando um pool de Trichoderma asperellum (T-06, T-09, T-12, e T-52), aplicados isoladamente ou em consórcio, mais o tratamento controle, sem aplicação de microrganismos. Os tratamentos mais efetivos foram os mixes de microrganismos 1301 + Ab-V5 e BRM 32114 + pool de Trichoderma asperellum, uma vez que proporcionaram aumento, em média, de 123% e 88% no número de panículas e de 206 e 167% na produtividade de grão do arroz de terras altas, respectivamente. Os consórcios 1301 + Ab-V5, BRM 32114 + pool de Trichoderma asperellum, BRM 32110 + BRM 32114, BRM 32110 + Ab-V5, 1301 + BRM 32110 e 1381 + pool de Trichoderma asperellum, também proporcionaram maior desempenho morfofisiológico nas plantas de arroz (taxa fotossintética, eficiência de carboxilação, número de perfilhos, biomassa seca de parte aérea e teor de nutrientes na parte aérea e raiz das plantas). Portanto, o uso de microrganismos multifuncionais no manejo do arroz de terras altas foi eficiente em sua capacidade de proporcionar maior desenvolvimento das plantas.

Statistical experimental designs were used to optimize conditions for recombinant Burkholderia pyrrocinia feruloyl esterase (BpFae) production in bacteria under lactose induction. After optimization by single factor design, Plackett–Burman design, steepest ascent design and the response surface method, the optimal conditions for BpFae production were: 6 g/L lactose, pH 5.5, pre-induced period 5 h, 23 °C, shaker rotational speed of 240 rpm, medium volume of 50 mL/250 mL, inoculum size 0.2% (v/v), and a post-induced period of 32 h in a Luria–Bertani culture. The produced BpFae activity was 7.43 U/mL, which is 2.92 times higher than that obtained under optimal conditions using IPTG as the inducer. BpFae activity was 4.82 U/mL in a 5 L fermenter under the abovementioned optimal conditions. BpFae produced a small amount of ethyl acetate but had no effect on the synthesis of other important esters in Baijiu. The results underpin further investigations into BpFae characterization and potential applications.

Introduction Burkholderia cepacia (B. cepacia) complex is composed of 20 phylogenetically closely related bacterial species. Some species have emerged as opportunistic pathogens in immunocompromised patients and are responsible for nosocomial outbreaks. The B. cepacia complex is a recognised respiratory pathogen in patients with cystic fibrosis. Burkholderia cenocepacia and Burkholderia multivorans (B. multivorans) are the most prevalent species in the world, according to the literature. However, research groups in Argentina have described a particular local epidemiology, with prevalence of Burkholderia contaminans (B. contaminans). Methods A total of 68 isolates of B. cepacia complex recovered of 46 cystic fibrosis patients attended at 14 hospitals distributed in 9 provinces of the country were studied. Identification was carried out by conventional phenotypic methods and was confirmed by recA gene sequencing. Sequences were analysed using the BLASTN program and comparing with B. cepacia complex type strains sequences deposited in GenBank. Antibiotic susceptibility tests were performed on isolates of the most prevalent species according to CLSI M45 guidelines. Results The prevalent specie was B. contaminans (49%, n = 33) followed by B. cenocepacia (25%; n = 17). The remaining species were Burkholderia seminalis (B. seminalis) (7%, n = 5), B. cepacia (7%, n = 5), B. multivorans (6%, n = 4), Burkholderia vietnamensis (5%, n = 3) and Burkholderia pyrrocinia (1%; n = 1). The 46% of B. contaminans isolates were resistant to SXT and 76% sensitive to MIN, MEM and CAZ. The isolates of B. cenocepacia were 100% resistant to SXT and MIN and 47% to CAZ and MEM. B. seminalis showed high levels of resistance to TMS (80%), CAZ (60%) and MIN (60%), and 60% of the isolates showed intermediate sensitivity to MEM. Conclusion Previous reports have described the prevalence of B. contaminans isolation from cystic fibrosis patients in Argentina, Spain and Portugal, and a case of two patients with cystic fibrosis in Ireland has recently been reported. Due to the high frequency with which B. contaminans is isolated in our country, it is necessary to promote the investigation of possible sources of infection and to understand the factors and mechanisms involved in the apparent greater transmissibility of this species. Different antimicrobial resistance profiles were detected between the species.

The use of beneficial microorganisms in forage grasses is a potentially advantageous technique for a more sustainable pasture management by decreasing the need for chemical fertilization. Our aims were to determine the best method of microorganism inoculation on Brachiaria (Syn. Urochloa) brizantha cv. BRS Piata, compare the responses of inoculated plants of this forage grass with fertilized and unfertilized controls and examine its effect on some morphological, physiological and biochemical responses. On the first experiment, three inoculation methods were tested: in the seed, seed and soil, and soil, with Pseudomonas fluorescens (BRM-32111) and Burkholderia pyrrocinia (BRM-32113). In the second experiment, fertilized and unfertilized plants were either inoculated with BRM-32111, BRM-32113 and co-inoculated (BRM-32111 + BRM-32113). In a final experiment, B. brizantha was inoculated by soil drenching with BRM-32111, BRM-32113 and co-inoculated (BRM-32111 + BRM-32113), and compared to fertilized- and unfertilized-controls. The inoculation by soil drenching, at seedling stage, was more effective than inoculation only in the seed or both in the seed and by soil drenching. The fertilizer may have suppressed the beneficial bacterial effects on the growth of B. brizantha. P. fluorescens and B. pyrrocinia co-inoculated increased nitrate, protein, nitrogen concentration, Spad index (chlorophyll content), leaf area, number of tillers, net photosynthesis and total biomass production of B. brizantha plants. Our results point out to a potentially valuable source of practical information in the search of an eco-friendlier approach to increase pasture productivity.

To expand the usage of endophytes in agriculture and in forestry, the insecticidal gene cry218 of Bacillus thuringiensis was introduced into a poplar bacterial endophyte Burkholderia pyrrocinia JK-SH007. The cry218 gene was cloned by polymerase chain reaction (PCR) and was inserted into a PHKT2 expression vector that was introduced into the bacterial endophyte JK-SH007. By using sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and western blotting, we confirmed that the engineered bacterial endophyte was successfully constructed, and it harboured insecticidal function after the bioassay in planta. The toxicity of the expressed insecticidal protein was analysed on second instar silkworm. The regression equation showed that the median lethal concentration (LC50) of the insecticidal protein was 0.77 (0.57–1.04) g/L at 72 h. The insecticidal bacteria genetically modified in this study have laid the foundation for further exploitation of biocontrol bacteria.

Burkholderia pyrrocinia JK-SH007 is a plant growth-promoting bacteria (PGPB), that can promote the growth of poplar and other trees, and, production of the plant hormone indole-3-acetic acid (IAA) is one of the reasons for this effect. Therefore, the aims of this study were to evaluate the effect of the external environment on the synthesis of IAA by B. pyrrocinia JK-SH007 and to perform a functional analysis of its IAA synthesis pathway. In this study, IAA and its synthetic intermediates indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), tryptamine (TAM), and indole-3-acetonitrile (IAN) were detected in B. pyrrocinia JK-SH007 fermentation broth by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS), and these indolic compounds were also found in the cell-free extraction of B. pyrrocinia JK-SH007, but the genomic analysis of B. pyrrocinia JK-SH007 indicated that IAA biosynthesis was mainly through the IAM and TAM pathways. The effects of L-tryptophan (L-Trp), temperature and pH on the synthesis of IAA were investigated, and the results showed that L-Trp exerted a significant effect on IAA synthesis and that 37°C and pH 7 were the optimal conditions IAA production by B. pyrrocinia JK-SH007. In addition, the protein expression of tryptophan 2-monooxygenase and indoleacetamide hydrolase, which are the key enzymes of the indole acetamide-mediated IAA synthesis pathway, was analyzed, and their activity was verified by substrate feeding experiments. The results revealed the existence of an IAA synthesis pathway mediated by IAM and indicated that this pathway plays a role in B. pyrrocinia JK-SH007. This study lays the foundation for further exploration of the specific pathway and mechanism of IAA synthesis in B. pyrrocinia JK-SH007.

Burkholderia pyrrocinia JK-SH007 is an important biocontrol strain for the prevention and treatment of poplar canker disease. Its powerful biocontrol function is inseparable from its successful colonization of poplar trees. Bacterial biofilms can ensure the long-term colonization of a host. To explore the mechanism of action of biofilms in the biocontrol process, we manipulated various exogenous factors to explore the morphology of the JK-SH007 biofilm in vitro. The addition of glycerol and MgSO4 to TSB medium stimulated biofilm production, increased the resistance of JK-SH007 to disease, enhanced the survival of JK-SH007 in nutrient-poor environments and maintained the antagonistic ability of JK-SH007 against the poplar canker pathogen. Therefore, we constructed and optimized a biofilm-forming system to produce a large number of stable JK-SH007 biofilms. The optimized system showed that the optimal incubation time for JK-SH007 biofilm formation was 14 h, the optimal temperature of the static culture was 25 °C, and the optimal pH was 5. The optimal medium for biofilm formation was TSB medium, 1% glycerol and 50 mM MgSO4. RT-qPCR experiments showed that an increase in the expression of the suhB gene promoted JK-SH007 biofilm formation, while an increase in the expression level of the ropN gene inhibited JK-SH007 biofilm formation. The possible mechanism by which JK-SH007 was inhibited by biofilm formation under natural culture was revealed. These results indicate the importance of adding nutrients to JK-SH007 biocides produced on a commercial scale. This is the first report of JK-SH007 producing a long-lasting biofilm that guarantees antagonism.