Environmental interactions are regulated by temperature in Burkholderia seminalis TC3.4.2R3.

Burkholderia seminalis strain TC3.4.2R3 is an endophytic bacterium isolated from sugarcane roots that produces antimicrobial compounds, facilitating its ability to act as a biocontrol agent against phytopathogenic bacteria. In this study, we investigated the thermoregulation of B. seminalis TC3.4.2R3 at 28 °C (environmental stimulus) and 37 °C (host-associated stimulus) at the transcriptional and phenotypic levels. The production of biofilms and exopolysaccharides such as capsular polysaccharides and the biocontrol of phytopathogenic fungi were enhanced at 28 °C. At 37 °C, several metabolic pathways were activated, particularly those implicated in energy production, stress responses and the biosynthesis of transporters. Motility, growth and virulence in the Galleria mellonella larvae infection model were more significant at 37 °C. Our data suggest that the regulation of capsule expression could be important in virulence against G. mellonella larvae at 37 °C. In contrast, B. seminalis TC3.4.2R3 failed to cause death in infected BALB/c mice, even at an infective dose of 107 CFU.mL-1. We conclude that temperature drives the regulation of gene expression in B. seminalis during its interactions with the environment

Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source

Endophytic fungi live inside plants, apparently do not cause any harm to their hosts and may play important roles in defense and growth promotion. Fungal growth is a routine practice at microbiological laboratories, and the Potato Dextrose Agar (PDA) is the most frequently used medium because it is a rich source of starch. However, the production of potatoes in some regions of the world can be costly. Aiming the development of a new medium source to tropical countries, in the present study, we used leaves from the guarana (a tropical plant from the Amazon region) and the olive (which grows in subtropical and temperate regions) to isolate endophytic fungi using PDA and Manihot Dextrose Agar (MDA). Cassava (Manihot esculenta) was evaluated as a substitute starch source. For guarana, the endophytic incidence (EI) was 90% and 98% on PDA and MDA media, respectively, and 65% and 70% for olive, respectively. The fungal isolates were sequenced using the ITS- rDNA region. The fungal identification demonstrated that the isolates varied according to the host plant and media source. In the guarana plant, 13 fungal genera were found using MDA and six were found using PDA. In the olive plant, six genera were obtained using PDA and 4 were obtained using MDA. The multivariate analysis results demonstrated the highest fungal diversity from guarana when using MDA medium. Interestingly, some genera were isolated from one specific host or in one specific media, suggesting the importance of these two factors in fungal isolation specificity. Thus, this study indicated that cassava is a feasible starch source that could serve as a potential alternative medium to potato medium.This work was supported by a grant from the Foundation for Research Assistance, São Paulo State and Amazon State, Brazil (grant n. 2009/53376-2) and by the National Council for Scientific and Technological Development (CNPq). We thank FAPESP for the M.C.Q. (grant no. 2010/50445-0), J.M. (grant no. 2011/18740-5) and S.T. (grant no. 2010/15192-4) and CNPq for E.F.S. and D.M.L. fellowships

Study of the n-TASE cluster on environmental interactions of B. seminalis TC3.4.2R3.

Espécies do gênero Burkholderia são fortemente associadas ao seu comportamento patogênico, devido ao agravamento do quadro de pacientes com o sistema imunológico comprometido. Apesar disso, existe um grande interesse nas bactérias desse gênero visando aplicações biotecnológicas, principalmente na área agrícola. Existem estudos que demonstram a importância de Burkholderia spp. na promoção de crescimento vegetal, biorremediação, controle biológico etc. A espécie Burkholderia seminalis tem sido isolada de diversos tipos de amostras como água, solo, rizosfera, sementes, plantas a clínicas, onde pode estar envolvida em infecções nosocomiais. Também é considerada fitopatogênica para damasco, em diversos modelos animais foi observado que essa espécie possui fraca virulência e, não apresenta capacidade de colonizar/infectar pulmões de ratos. A linhagem TC3.4.2R3 de B. seminalis, isolada das raízes de cana-de-açúcar, apresenta atividade antagônica frente a uma série de fungos fitopatogênicos. Além disso, apresenta uma atividade potencial para o controle de sintomas de necrose foliar em orquídeas causada pela espécie Burkholderia gladioli. Algumas características relacionadas à promoção de crescimento foram observadas nessa linhagem. A linhagem TC3.4.2R3 possui uma região que compreende às locus tags Bsem_02857 a Bsem_02860 (cluster n-TASE), ausente nos demais genomas de Burkholderia utilizados como referência para sua anotação (B. cenocepacia J2315 e B. ambifaria AMMD). Acredita-se que esse cluster tenha sido adquirido por transferência horizontal e mantido no genoma desta bactéria por pressão de seleção. Assim, o principal objetivo do presente estudo foi entender a origem evolutiva desse cluster gênico, bem como o seu papel nos mecanismos de interação em TC3.4.2R3. Avaliou-se a história evolutiva do cluster n-TASE em TC3.4.2R3, por meio da combinação de métodos de estudo de HGT como filogenia, genômica comparativa e análise molecular. Posteriormente, foi realizada a caracterização do cluster n-TASE, por meio da obtenção de dois mutantes via mutagênese insercional com o plasmídeo pGP&#937Tp, SST57 com mutação no gene Bsem_02857 e SST28 com mutação no gene Bsem_02858. O cluster n-TASE de TC3.4.2R3 apresentou maior similaridade com as sequências homólogas de Aquamicrobium sp. SK-2 e B. contaminans LMG23361. Foi possível fornecer evidências de que o n-TASE foi adquirido por meio de um evento de HGT Observou-se uma correlação de natureza ainda desconhecida dos genes do n-TASE cluster com motilidade do tipo swarming, produção de biofilme, tolerância ao estresse oxidativo e sobrevivência bacteriana em hemolinfa de G. mellonella, mas não correlacionada à virulência bacteriana a este tipo de modelo de infecção. Futuramente, serão necessários experimentos para o melhor entendimento da forma como esses genes atuam nesses processos.Species of the Burkholderia genus are strongly associated with its pathogenic behavior, due to the worsening of patients with compromised immune systems. Despite this, there is a great interest in bacteria of this genus aiming at biotechnological applications, mainly in the agricultural area. There are studies that demonstrate the importance of Burkholderia spp. in the promotion of plant growth, bioremediation, biological control etc. The Burkholderia seminalis species has been isolated from different types of samples such as water, soil, rhizosphere, seeds, plants and clinics, where it may be involved in nosocomial infections. It is also considered phytopathogenic for apricots, in several animal models it was observed that this species has weak virulence and does not show the ability to colonize/infect rat lungs. The TC3.4.2R3 strain of B. seminalis, isolated from sugarcane roots, presents antagonistic activity against a series of phytopathogenic fungi. Furthermore, it has a potential activity to control symptoms of leaf necrosis in orchids caused by Burkholderia gladioli species. Some characteristics related to growth promotion were observed in this strain. The TC3.4.2R3 lineage has a region comprising the locus tags Bsem_02857 to Bsem_02860 (cluster n-TASE), absent in the other Burkholderia genomes used as reference for its annotation (B. cenocepacia J2315 and B. ambifaria AMMD). It is believed that this cluster was acquired by horizontal transfer and maintained in the genome of this bacterium by selection pressure. Thus, the main objective of this study was to understand the evolutionary origin of this gene cluster, as well as its role in the interaction mechanisms in TC3.4.2R3. The evolutionary history of the n-TASE cluster in TC3.4.2R3 was evaluated by combining HGT study methods such as phylogeny, comparative genomics and molecular analysis. Subsequently, the characterization of the n-TASE cluster was performed, by obtaining two mutants via insertional mutagenesis with the plasmid pGP&#937Tp, SST57 with mutation in the Bsem_02857 gene and SST28 with mutation in the Bsem_02858 gene. The n-TASE cluster of TC3.4.2R3 showed greater similarity with the homologous sequences of Aquamicrobium sp. SK-2 and B. contaminans LMG23361. It was possible to provide evidence that n-TASE was acquired through an HGT event. A correlation of a still unknown nature of the n-TASE cluster genes with swarming motility, biofilm production, tolerance to oxidative stress and bacterial survival in G. mellonella hemolymph, but not correlated to bacterial virulence in this type of infection model. In the future, experiments will be needed to better understand how these genes act in these processes

Deciphering the transposable elements in Puccinia psidii Winter, causal agent of rust on Eucalyptus spp.

A cultura do eucalipto apresenta grande importância no setor florestal no mundo. No Brasil, 70% da área florestal plantada é destinada ao eucalipto. Entretanto, a ferrugem das mirtáceas, também conhecida como ferrugem do eucalipto, causada pelo fungo Puccinia psidii Winter, afeta o enorme potencial produtivo das plantações de eucalipto. A biologia, mecanismos de patogenicidade e genética desse patógeno são pouco conhecidos, apesar de sua importância para o setor florestal. Os elementos transponíveis (TEs) são sequências de DNA com a capacidade de migrar e influenciar a organização, integridade e evolução do genoma hospedeiro. O presente trabalho teve como principal objetivo estudar os TEs presentes no genoma de P. psidii, combinando ferramentas in silico e moleculares. A classificação dos elementos transponíveis no genoma de P. psidii MF-1 foi realizada utilizando contigs previamente minerados e remontados, bem como sem seleção prévia dos contigs, por meio do programa RepeatMasker. Ambas estratégias apontaram o predomínio de elementos da Classe I - LTR Retrotransposons no genoma de P. psidii MF-1. O resultado condiz com a composição de TEs em fungos fitopatogênicos descrita na literatura. Algumas análises in silico, como verificação de integridade e anotação manual de sequências proteicas foram também realizadas para alguns contigs classificados como TEs. Assim, foi possível observar a presença de sequências conservadas pertencentes à região pol em LTR Retrotransposons. Além disso, as análises permitiram inferir sobre a existência de TEs híbridos no genoma parcialmente sequenciado de P. psidii MF-1. Paralelamente foi também realizada uma análise comparativa entre os TEs presentes nos genomas de P. graminis, P. striiformis, P. triticina e P. psidii. Observou-se que P. graminis, P. striiformis e P. triticina apresentam maior frequência de elementos da Classe II, do tipo DNA Transposons ao contrário de P. psidii, com maior frequência de elementos da Classe I. Interessantemente, a quantidade de elementos desconhecidos foi similarmente alta para todos os quatro genomas avaliados. Este tipo de análise é muito importante, pois evidencia a grande quantidade de famílias de TEs novas a serem descobertas. Elas podem estar potencialmente relacionadas ao silenciamento de genes importantes à virulência destes patógenos. A utilização de TEs no estudo de diversidade genética entre populações é bastante comum. A técnica molecular IRAP foi utilizada para acessar a diversidade entre populações de P. psidii originárias de três híbridos de Eucalyptus spp., goiabeira, jambeiro e jabuticabeira. No entanto, esta técnica não se mostrou eficiente para detectar polimorfismos existentes entre estas populações. A anotação de TEs foi difícil devido à observação de sequências de elementos sobrepostas, o que podem representar híbridos de TEs, entretanto, visando a confirmação desta hipótese por meio da PCR, alguns contigs serão sequenciados e mais estudos devem ser realizados para a continuação desta confirmação. Os resultados apresentados neste trabalho são inéditos e representam uma etapa crucial no entendimento de TEs em fungos do gênero Puccinia, em especial do patógeno P. psidii para o desenvolvimento de melhores mecanismos de controle de ferrugem.The culture of eucalyptus has great importance worldwide in forestry sector. In Brazil, 70% of cultivated forest area is intended for Eucalyptus. However, the eucalyptus potential productive has been affected by rust disease, caused by the fungus Puccinia psidii Winter. Despite its importance to brazilian and world forest sector, the knowledge of biology, genetic and pathogenic mechanisms of this pathogen is scarce. Transposable elements (TEs) are mobile DNA fragments that influence the organization and development of the host genome. These elements have the ability to move within host genome, and their insertion can cause a wide spectrum of mutations in their hosts. This study aims to decipher the TEs in P. psidii genome by combining in silico and molecular tools. P. psidii MF-1 TEs classification was performed automatically, through RepeatMasker software, being observed a predominance of Class I - LTR Retrotransposons in P. psidii MF-1 genome. This result is consistent with the TEs composition described in phytopathogenic fungi. Some in silico analysis, as integrity and manual annotation of conserved protein sequences from TEs were carried out with P. psidii MF-1 contigs classified as transposable elements. The presence of conserved sequences belonging to pol region in LTR Retrotransposons was observed. Furthermore, these analysis allowed the inference of hybrid TEs in P. psidii MF-1. At the same time, a comparative analysis of TEs present in other Puccinia genomes and P. psidii MF-1 was also performed. The P. graminis, P. striiformis and P. triticina genomes have higher frequency of Class II - DNA Transposons unlike the results found for P. psidii. Interestingly, the number of unknown elements was similarly high for all genomes. This type of analysis is very importante because it shows a great number of potential new TEs families to be discovered. They may be potentially related to the virulence gene silencing of these pathogens. Using TEs for study the fungal genetic diversity is quite common. The IRAP technique was used to access the diversity among P. psidii populations originated from three Eucalyptus spp. hybrids, guava, syzigium and jabuticaba. However, this technique was not efficient to detect existing polymorphisms between these populations. TEs annotation was labored due to the existence of overlapping elements, which may represent hybrids TEs. PCR tool was used to confirm some sequences annotated as hybrids and more studies are needed to confirm this hyphotesis. The results presented in this study are novel and is a crucial step in understanding the genetic of P. psidii pathogen for further improvements of rust control mechanisms

A look into a multifunctional toolbox: endophytic Bacillus species provide broad and underexploited benefits for plants

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

Antagonistic activity of fungi from anthracnose lesions on Paullinia cupana against Colletotrichum sp.

Anthracnose is a cosmopolitan disease caused by Colletotrichum spp. that affects many crops worldwide. Observations have shown that anthracnose leaf lesions may be colonized by several non-pathogenic microorganisms. The relationship of these microorganisms with the pathogen as well as their potential as biocontrol agents is not well known. Guarana (Paullinia cupana Mart. var. sorbilis) is a typical native Amazon crop with unknown microbial diversity. Guarana productivity has been reduced by a fungal disease caused by Colletotrichum sp. In this study, we isolated 15 fungi from guarana anthracnose leaf lesions that belong to five genera: Fusarium, Phomopsis, Leptosphaeria, Microdochium and Pestalotiopsis. Four isolates from the Fusarium sp. (C6 and C10), Pestalotiopsis sp. (C3), and Microdochium sp. (P7) consistently inhibited anthracnose fungal growth in vitro. Except for the Microdochium sp. (P7), these isolates were also able to inhibit the growth of the pathogen in in vivo assays using detached guarana leaves. Some mechanisms related to the growth inhibition of this pathogen were studied. Fusarium sp. (C6) produced chitinases; Fusarium sp. (C6, C10) and Pestalotiopsis sp. (C3) produced antagonistic volatile organic compounds. These three isolates also inhibited the growth of Fusarium spp., a pathogen of several plant species, suggesting their potential broad range of growth inhibition of other phytopathogens

Bacterial communities associated with anthracnose symptomatic and asymptomatic leaves of guarana, an endogenous tropical crop, and their pathogen antagonistic efects

Plants are colonized by diverse microorganisms that can substantially impact their health and growth. Understanding bacterial diversity and the relationships between bacteria and phytopathogens may be key to finding effective biocontrol agents. We evaluated the bacterial community associated with anthracnose symptomatic and asymptomatic leaves of guarana, a typical tropical crop. Bacterial communities were assessed through culture-independent techniques based on extensive 16S rRNA sequencing, and cultured bacterial strains were evaluated for their ability to inhibit the growth of Colletotrichum sp. as well as for enzyme and siderophore production. The culture-independent method revealed that Proteobacteria was the most abundant phylum, but many sequences were unclassified. The emergence of anthracnose disease did not significantly affect the bacterial community, but the abundance of the genera Acinetobacter, Pseudomonas and Klebsiella were significantly higher in the symptomatic leaves. In vitro growth of Colletotrichum sp. was inhibited by 11.38% of the cultured bacterial strains, and bacteria with the highest inhibition rates were isolated from symptomatic leaves, while asymptomatic leaves hosted significantly more bacteria that produced amylase and polygalacturonase. The bacterial isolate Bacillus sp. EpD2-5 demonstrated the highest inhibition rate against Colletotrichum sp., whereas the isolates EpD2-12 and FD5-12 from the same genus also had high inhibition rates. These isolates were also able to produce several hydrolytic enzymes and siderophores, indicating that they may be good candidates for the biocontrol of anthracnose. Our work demonstrated the importance of using a polyphasic approach to study microbial communities from plant diseases, and future work should focus on elucidating the roles of culture-independent bacterial communities in guarana anthracnose disease

Screening of tropically derived, multi-trait plant growth- promoting rhizobacteria and evaluation of corn and soybean colonization ability

The present study assessed the plant growth-promoting (PGP) traits and diversity of culturable rhizobacteria associated with guarana (Paullinia cupana), a typical tropical plant. Ninety-six bacteria were isolated, subjected to biochemical tests, and identified by partial or total 16S rDNA sequencing. Proteobacteria and Firmicutes were the dominant rhizospheric phyla found, and Burkholderia and Bacillus were the most abundant genera. Thirteen strains exhibited the four PGP traits evaluated, and most of them belonged to the genus Burkholderia. Two multitrait PGP strains, RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia ambifaria), expressively promoted corn and soybean growth under greenhouse conditions. Compared to the non-inoculated control, increases in corn root dry weight of 247.8 and 136.9% were obtained with RZ2MS9 and RZ2MS16 inoculation, respectively, at 60 days after seeding. The dry weights of corn and soybean shoots were significantly higher than those of non-inoculated plants, showing increases of more than 47% for both strains and crops. However, soybean root dry weight did not increased after bacterial inoculation with either strain. The colonization behavior of RZ2MS16 was assessed using GFP-labeling combined with fluorescence microscopy and a cultivation-based approach for quantification. RZ2MS16:gfp was able to colonize the roots and shoots of corn and soybean, revealing an endophytic behavior