Mycoviruses infecting Colletotrichum spp: a comprehensive review / Micoviroses em Colletotrichum spp: uma revisão abrangente

Colletotrichum is one of the most economically important fungal genera, which affects a wide range of hosts, specifically tropical and subtropical crops. Thus far, there have been several records of mycovirus infection in Colletotrichum spp., primarily by viruses of the Partitiviridae family. There have also been records of infections by mycoviruses of the Chrysoviridae family. Mycoviruses are (+)ssRNA and dsRNA genome viruses, which may or may not be enveloped. To date, no mycovirus with a DNA genome has been isolated from Colletotrichum spp. Typically, mycoviruses cause latent infections, although hypo- and hypervirulence have also been reported in Colletotrichum spp. In addition to its effects on pathogenic behavior, mycovirus infection can lead to important physiological changes, such as altered morphological characteristics, reduced vegetative growth, and suppressed conidia production. Therefore, research on mycoviruses infecting phytopathogenic fungi can help develop alternative methods to chemical control, which can cause irreversible damage to humans and the environment. From an agricultural perspective, mycoviruses can contribute to sustainable agriculture as biological control agents via changes in fungal physiology, ultimately resulting in the total loss of or reduction in the virulence of these pathogens.

Colletotrichum é um dos gêneros fúngicos mais importantes economicamente, afetando uma ampla gama de hospedeiros, especialmente em cultivos tropicais e subtropicais. Atualmente já existem diversos registros de infecção por micovírus em Colletotrichum spp., sendo a maioria dos já identificados classificados na família Partitiviridae. Ocorrem registros também de micovírus pertencentes à família Chrysoviridae. Compreendem vírus de genoma de (+)ssRNA e dsRNA que podem ser ou não envelopados. Ainda não foram identificados micovírus com genoma de DNA isolados de Colletotrichum. A infecção por micovírus pode ocorrer de forma latente, mas já foi observado em Colletotrichum spp. o fenômeno de hipo e hipervirulência. Além de influenciar no comportamento patogênico, a infecção pode causar mudanças fisiológicas importantes como alterações das características morfológicas, redução do crescimento vegetativo e redução na produção de conídios. O estudo com micovírus em fungos fitopatogênicos traz uma alternativa ao controle químico que é um método capaz de causar danos irreversíveis ao homem e o meio ambiente. Sob a perspectiva agrícola, os micovírus podem contribuir para agricultura sustentável como agentes de controle biológico. Isso porque obsevam-se mudanças importantes na fisiologia fúngica resultando na perda total ou redução da virulência desses patógenos.

Mycoviruses infecting Colletotrichum spp.: A comprehensive review

Abstract Colletotrichum is one of the most economically important fungal genera, which affects a wide range of hosts, specifically tropical and subtropical crops. Thus far, there have been several records of mycovirus infection in Colletotrichum spp., primarily by viruses of the Partitiviridae family. There have also been records of infections by mycoviruses of the Chrysoviridae family. Mycoviruses are (+)ssRNA and dsRNA genome viruses, which may or may not be enveloped. To date, no mycovirus with a DNA genome has been isolated from Colletotrichum spp. Typically, mycoviruses cause latent infections, although hypo- and hypervirulence have also been reported in Colletotrichum spp. In addition to its effects on pathogenic behavior, mycovirus infection can lead to important physiological changes, such as altered morphological characteristics, reduced vegetative growth, and suppressed conidia production. Therefore, research on mycoviruses infecting phytopathogenic fungi can help develop alternative methods to chemical control, which can cause irreversible damage to humans and the environment. From an agricultural perspective, mycoviruses can contribute to sustainable agriculture as biological control agents via changes in fungal physiology, ultimately resulting in the total loss of or reduction in the virulence of these pathogens.

Resumo Colletotrichum é um dos gêneros fúngicos mais importantes economicamente, afetando uma ampla gama de hospedeiros, especialmente em cultivos tropicais e subtropicais. Atualmente já existem diversos registros de infecção por micovírus em Colletotrichum spp., sendo a maioria dos já identificados classificados na família Partitiviridae. Ocorrem registros também de micovírus pertencentes à família Chrysoviridae. Compreendem vírus de genoma de (+)ssRNA e dsRNA que podem ser ou não envelopados. Ainda não foram identificados micovírus com genoma de DNA isolados de Colletotrichum. A infecção por micovírus pode ocorrer de forma latente, mas já foi observado em Colletotrichum spp. o fenômeno de hipo e hipervirulência. Além de influenciar no comportamento patogênico, a infecção pode causar mudanças fisiológicas importantes como alterações das características morfológicas, redução do crescimento vegetativo e redução na produção de conídios. O estudo com micovírus em fungos fitopatogênicos traz uma alternativa ao controle químico que é um método capaz de causar danos irreversíveis ao homem e o meio ambiente. Sob a perspectiva agrícola, os micovírus podem contribuir para agricultura sustentável como agentes de controle biológico. Isso porque obsevam-se mudanças importantes na fisiologia fúngica resultando na perda total ou redução da virulência desses patógenos.

Mycoviruses infecting Colletotrichum spp: A comprehensive review / Micoviroses em Colletotrichum spp: uma revisão abrangente

Abstract Colletotrichum is one of the most economically important fungal genera, which affects a wide range of hosts, specifically tropical and subtropical crops. Thus far, there have been several records of mycovirus infection in Colletotrichum spp., primarily by viruses of the Partitiviridae family. There have also been records of infections by mycoviruses of the Chrysoviridae family. Mycoviruses are (+)ssRNA and dsRNA genome viruses, which may or may not be enveloped. To date, no mycovirus with a DNA genome has been isolated from Colletotrichum spp. Typically, mycoviruses cause latent infections, although hypo- and hypervirulence have also been reported in Colletotrichum spp. In addition to its effects on pathogenic behavior, mycovirus infection can lead to important physiological changes, such as altered morphological characteristics, reduced vegetative growth, and suppressed conidia production. Therefore, research on mycoviruses infecting phytopathogenic fungi can help develop alternative methods to chemical control, which can cause irreversible damage to humans and the environment. From an agricultural perspective, mycoviruses can contribute to sustainable agriculture as biological control agents via changes in fungal physiology, ultimately resulting in the total loss of or reduction in the virulence of these pathogens.

Resumo Colletotrichum é um dos gêneros fúngicos mais importantes economicamente, afetando uma ampla gama de hospedeiros, especialmente em cultivos tropicais e subtropicais. Atualmente já existem diversos registros de infecção por micovírus em Colletotrichum spp., sendo a maioria dos já identificados classificados na família Partitiviridae. Ocorrem registros também de micovírus pertencentes à família Chrysoviridae. Compreendem vírus de genoma de (+)ssRNA e dsRNA que podem ser ou não envelopados. Ainda não foram identificados micovírus com genoma de DNA isolados de Colletotrichum. A infecção por micovírus pode ocorrer de forma latente, mas já foi observado em Colletotrichum spp. o fenômeno de hipo e hipervirulência. Além de influenciar no comportamento patogênico, a infecção pode causar mudanças fisiológicas importantes como alterações das características morfológicas, redução do crescimento vegetativo e redução na produção de conídios. O estudo com micovírus em fungos fitopatogênicos traz uma alternativa ao controle químico que é um método capaz de causar danos irreversíveis ao homem e o meio ambiente. Sob a perspectiva agrícola, os micovírus podem contribuir para agricultura sustentável como agentes de controle biológico. Isso porque obsevam-se mudanças importantes na fisiologia fúngica resultando na perda total ou redução da virulência desses patógenos.

Mycoviruses infecting Colletotrichum spp.: A comprehensive review.

Colletotrichum is one of the most economically important fungal genera, which affects a wide range of hosts, specifically tropical and subtropical crops. Thus far, there have been several records of mycovirus infection in Colletotrichum spp., primarily by viruses of the Partitiviridae family. There have also been records of infections by mycoviruses of the Chrysoviridae family. Mycoviruses are (+)ssRNA and dsRNA genome viruses, which may or may not be enveloped. To date, no mycovirus with a DNA genome has been isolated from Colletotrichum spp. Typically, mycoviruses cause latent infections, although hypo- and hypervirulence have also been reported in Colletotrichum spp. In addition to its effects on pathogenic behavior, mycovirus infection can lead to important physiological changes, such as altered morphological characteristics, reduced vegetative growth, and suppressed conidia production. Therefore, research on mycoviruses infecting phytopathogenic fungi can help develop alternative methods to chemical control, which can cause irreversible damage to humans and the environment. From an agricultural perspective, mycoviruses can contribute to sustainable agriculture as biological control agents via changes in fungal physiology, ultimately resulting in the total loss of or reduction in the virulence of these pathogens.

Extracellular enzymatic profiles and taxonomic identification of endophytic fungi isolated from four plant species.

Plants of medicinal and economic importance have been studied to investigate the presence of enzyme-producing endophytic fungi. The characterization of isolates with distinct enzyme production potential may identify suitable alternatives for specialized industry. At Universidade Estadual de Maringá Laboratory of Microbial Biotechnology, approximately 500 isolates of endophytic fungi have been studied over the last decade from various host plants, including medicinally and economically important species, such as Luehea divaricata (Martius et Zuccarini), Trichilia elegans A. Juss, Sapindus saponaria L., Piper hispidum Swartz, and Saccharum spp. However, only a fraction of these endophytes have been identified and evaluated for their biotechnological application, having been initially grouped by morphological characteristics, with at least one representative of each morphogroup tested. In the current study, several fungal strains from four plants (L. divaricata, T. elegans, S. saponaria, and Saccharum spp) were identified by ribosomal DNA typing and evaluated semi-quantitatively for their enzymatic properties, including amylase, cellulase, pectinase, and protease activity. Phylogenetic analysis revealed the presence of four genera of endophytic fungi (Diaporthe, Saccharicola, Bipolaris, and Phoma) in the plants examined. According to enzymatic tests, 62% of the isolates exhibited amylase, approximately 93% cellulase, 50% pectinase, and 64% protease activity. Our results verified that the composition and abundance of endophytic fungi differed between the plants tested, and that these endophytes are a potential enzyme production resource of commercial and biotechnological value.

Rhizosphere bacteriome of the medicinal plant Sapindus saponaria L. revealed by pyrosequencing.

Sapindus saponaria L. of Sapindaceae family is popularly known as soldier soap and is found in Central and South America. A study of such medicinal plants might reveal a more complex diversity of microorganisms as compared to non-medicinal plants, considering their metabolic potential and the chemical communication between their natural microbiota. Rhizosphere is a highly diverse microbial habitat with respect to both the diversity of species and the size of the community. Rhizosphere bacteriome associated with medicinal plant S. saponaria is still poorly known. The objective of this study was to assess the rhizosphere microbiome of the medicinal plant S. saponaria using pyrosequencing, a culture-independent approach that is increasingly being used to estimate the number of bacterial species present in different environments. In their rhizosphere microbiome, 26 phyla were identified from 5089 sequences of 16S rRNA gene, with a predominance of Actinobacteria (33.54%), Acidobacteria (22.62%), and Proteobacteria (24.72%). The rarefaction curve showed a linear increase, with 2660 operational taxonomic units at 3% distance sequence dissimilarity, indicating that the rhizosphere microbiome associated with S. saponaria was highly diverse with groups of bacteria important for soil management, which could be further exploited for agricultural and biotechnological purposes.

RNA applications for endophytic research.

Endophytic microorganisms, mainly bacteria and fungi, have intrinsic relationships with the host plants, involving complex chemical and genetic communication networks. The relationship among these organisms involves the development of regulatory mechanisms of gene expression that control their development and response to different interactions. Although RNA molecules are already being used in studies of microorganism diversity and taxonomy, for example, using comparisons of rRNA regions, they may also be useful tools in the exploration of gene regulation and modeling of other molecules, such as the analysis of microRNA and small interfering RNAs. Transcriptional profile analyses are capable of providing robust information on biosynthetic pathways, genes involved in the interaction and differential production of metabolites by endophytes, using RNA-seq approaches. In-depth studies of RNA types and their functions in endophytes may provide valuable information that can be used for biotechnological manipulation of microorganisms to produce metabolites, bioremediation, biological control of pathogens, and decrease plant diseases, among other economically important applications. Our study highlights the present state of knowledge of studies involving endophytes, RNA molecules, and future perspectives.

Agrobacterium-mediated transformation of Fusarium proliferatum.

Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent.

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.

Molecular phylogeny and biotechnological potential of bacterial endophytes associated with Malpighia emarginata.

Acerola (Malpighia emarginata) is a shrub native to tropical and subtropical climates, which has great commercial interest due to the high vitamin C content of its fruit. However, there are no reports of the endophytic community of this plant species. The aim of this study was to verify the genetic diversity of the leaf endophytic bacterial community of two varieties (Olivier & Waldy Cati 30) of acerola, and to evaluate their biotechnological ability by assessing their in vitro control of pathogenic fungi and the enzymatic production of cellulase, xylanase, amylase, pectinase, protease, lipase, esterase, and chitinase. In total, 157 endophytic bacteria were isolated from the leaves of two varieties of the plant at 28° and 37°C. Phylogenetic analysis confirmed the molecular identification of 58 bacteria, 39.65% of which were identified at the species level. For the first time, the genus Aureimonas was highlighted as an endophytic bacterium. Furthermore, 12.82% of the isolates inhibited the growth of all phytopathogens evaluated and at least one of the above-mentioned enzymes was produced by 64.70% of the endophytes, demonstrating that M. emarginata isolates have potential use in biotechnological studies.

Effects of IGF-1 on In Vitro Culture of Bovine Preantral Follicles are Dose-Dependent.

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF-1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α-MEM(+) , supplemented with different concentrations of human recombinant IGF-1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO2 in 24-well plates with total replacement of the medium every 2 days. Non-cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF-1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF-1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF-1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF-1 tested. Ultrastructurally, the non-cultured control and IGF-1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF-1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.

Investigation of mycoviruses in endophytic and phytopathogenic strains of Colletotrichum from different hosts.

Fungi belonging to the Colletotrichum genus can be categorized as endophytic or phytopathogenic. These fungi can be infected by viruses, termed mycoviruses, which are know to promote hypovirulence in infected fungi. However, there are few studies that have described mycoviral infections of endophytes. The production of secondary metabolites by endophytes with antimicrobial potential in inhibiting numerous pathogens has gained increasing attention. The aim of the current study was to investigate the presence of mycoviruses in endophytic and phytopathogenic fungi of the Colletotrichum genus, as well as to analyze the antimicrobial activity of crude extracts obtained from these samples. To detect the presence of mycoviruses in the samples, dsRNA was extracted, treated with enzymes, and analyzed following electrophoresis in agarose gel. Furthermore, isometric mycoviral particles were observed by transmission electron microscopy. Serial microdilution methodology was used to test crude extracts of Colletotrichum spp for antibacterial activity against Escherichia coli and Staphylococcus aureus, and antifungal activity against Fusarium solani. The results of the molecular and microscopic analyses indicated that a phytopathogenic strain presented infection by mycovirus. The antibacterial activity analysis revealed that the minimum inhibitory concentrations and minimum bactericidal concentrations were low for the fungal extracts of the two endophytes, indicating that these extracts were effective antibacterial agents. However, their antifungal activity against F. solani was not statistically different compared to that of the negative control.

In silico analysis of the 16S rRNA gene of endophytic bacteria, isolated from the aerial parts and seeds of important agricultural crops.

Because of human population growth, increased food production and alternatives to conventional methods of biocontrol and development of plants such as the use of endophytic bacteria and fungi are required. One of the methods used to study microorganism diversity is sequencing of the 16S rRNA gene, which has several advantages, including universality, size, and availability of databases for comparison. The objective of this study was to analyze endophytic bacterial diversity in agricultural crops using published papers, sequence databases, and phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S rRNA gene was used to identify endophytic bacteria, in important agricultural crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes, located in different geographical regions (America, Europe, and Asia). The corresponding 16S rRNA gene sequences were selected from the NCBI database, aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The most common orders present in the analyzed cultures were Bacillales, Enterobacteriales, and Actinomycetales and the most frequently observed genera were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that only approximately 1.56% of the total sequences were not properly grouped, demonstrating reliability in the identification of microorganisms. This study identified the main genera found in endophytic bacterial cultures from plants, providing data for future studies on improving plant agriculture, biotechnology, endophytic bacterium prospecting, and to help understand relationships between endophytic bacteria and their interactions with plants.

Biotechnological prospecting of foliar endophytic fungi of guaco (Mikania glomerata Spreng.) with antibacterial and antagonistic activity against phytopathogens.

Mikania glomerata (Spreng.), popularly known as "guaco", is a plant from the Asteraceae family that has many therapeutic properties. The use of medicinal plants has been examined in studies on endophytic diversity and bioprospecting; endophytes inhabit the interior of plants without harming them. Microorganism-host complex interactions are related to the production of compounds that may confer resistance to pathogens or to production of bioactive compounds or growth regulators. In this study, we evaluated foliar endophytic fungi of M. glomerata to examine the control of plant pathogens, molecular identification, and production of compounds with antimicrobial activity. In the antagonism test, 6-mm diameter disks were placed equidistant from the endophyte and plant pathogen, and pathogen growth area was measured. The endophytic strains G-01, G-02, and G-03 were effective against Fusarium solani and Didymella bryoniae. The endophyte rDNA regions corresponding to internal transcribed spacer 1-5.8S-internal transcribed spacer 2 were sequenced, and the results were compared with sequences deposited in the NCBI database. The G-01, G-02, and G-03 strains were identified as Diaporthe citri. This identification was confirmed by phylogenetic analysis. The crude extract of the secondary metabolites of the G-01 strain was tested against Escherichia coli and Staphylococcus aureus; the metabolites showed antimicrobial activity against S. aureus. The endophytes tested in this study have potential for use in biotechnological applications.

Molecular characterization of the endophytic fungal community associated with Eichhornia azurea (Kunth) and Eichhornia crassipes (Mart.) (Pontederiaceae) native to the Upper Paraná River floodplain, Brazil.

Endophytic fungi live in the interior of healthy plants without causing them any damage. These fungi are of biotechnological interest; they may be used in the biological control of pests and plant diseases, and in the pharmaceutical industry. The aquatic macrophytes Eichhornia azurea (Kunth) and Eichhornia crassipes (Mart.) belong to the Pontederiaceae family. The first is a fixed-floating species and the second is a free-floating species that is known for its phytoremediation potential. The fungal endophytes associated with the leaves of E. azurea and E. crassipes, native to the Upper Paraná River floodplain, Brazil, were isolated. The sequencing of the ITS1-5.8S-ITS2 region of ribosomal DNA was performed and the nucleotide sequences obtained were compared with those available in the GenBank database for the molecular identification of the isolates. The construction of phylogenetic trees was performed using the MEGA5 software. The results showed that high colonization frequencies were obtained from the 610 foliar fragments sampled from each plant: 87.86% for E. azurea and 88.85% for E. crassipes. At the genus level, it was possible to identify 19 fungal endophytes belonging to the genera Alternaria, Bipolaris, Cercospora, Diaporthe, Gibberella, Pestalotiopsis, Plectosphaerella, Phoma, and Saccharicola. Two other endophytes were identified at the species level (Microsphaeropsis arundinis). Genera Bipolaris, Cercospora, Microsphaeropsis, and Phoma were found as endophytes in the two macrophytes and the other genera were host-specific, being isolated from only one macrophyte, proving that there is a small difference in the endophytic diversity of the two Eichhornia species analyzed.

Genetic variability analysis of Zymomonas mobilis strains from the UFPEDA microorganisms collection.

Zymomonas mobilis is a Gram-negative bacterium that has drawn attention in the bioethanol industry. Besides bioethanol, this bacterium also produces other biotechnological products such as levans, which show antitumor activity. Molecular studies involving Z. mobilis have advanced to the point that allows us to characterize interspecies genetic diversity and understand their metabolism, and these data are essential for better utilization of this species. In this study, the genetic diversity of 24 strains from the Microorganisms Collection of Departamento de Antibióticos (UFPEDA) from Universidade Federal de Pernambuco were characterized. The methods used were amplified ribosomal DNA restriction analysis and diversity analysis of the internally transcribed 16S-23S rDNA spacer region (ISR). These analyses revealed low genetic variability of the 16S rDNA gene. These data confirm that these isolates are, or are closely related to, Z. mobilis. Moreover, the analysis of the ISR confirmed the genetic variability of strains deposited in the UFPEDA collection of microorganisms and grouped these strains into ten ribotypes, which can be used in the future for breeding programs and for the preservation of biodiversity. Furthermore, this study characterized the genetic variability between the UFPEDA 205/ ZAP, UFPEDA 98/AG11, and ZAG strains, which were obtained by spheroplast fusion among them. The data also indicate that there is genetic variability among the UFPEDA 202/CP4 and UFPEDA 633/ ZM4 strains, demonstrating that these important Z. mobilis strains are distinct, as suggested in previous studies.

Phylogenetic analysis of endophytic bacterial isolates from leaves of the medicinal plant Trichilia elegans A. Juss. (Meliaceae).

Various organisms such as fungi and bacteria can live inside plants, inhabiting the aerial parts (primarily the leaves) without causing damage. These microorganisms, called endophytes, produce an extensive variety of compounds that can be useful for medical and agronomic purposes. Trichilia elegans A. Juss., belonging to the Meliaceae family, shows wide dispersion in South America, and phytochemical analyses from these plants and endophyte isolates have shown biological activity. Accordingly, the aim of this study was to verify the diversity of bacterial endophytes from T. elegans using partial sequencing of 16S rRNA, followed by phylogenetic analysis. Isolation was performed by cutting the leaves, after disinfection with 5% sodium hypochlorite (NaOCl), in 1-2-mm² fragments, which were equally placed on dishes containing TSA and fungicide BENLATE at 75 µg/mL. All dishes were incubated at 28°C in the biochemical oxygen demand system for 5 days and periodically checked. Afterwards, the colonization frequency (%) was determined: (number of fragments colonized by bacteria/total number of fragments) x 100. Three isolations between September 2011 and March 2012 were performed; the growth frequency ranged between 1.6 and 13.6%. Following sequencing of 16S rRNA and phylogenetic analysis, the genera identified were: Staphylococcus, Bacillus, Microbacterium, Pseudomonas, and Pantoea. These results will provide important knowledge on the diversity of endophytic bacteria inhabiting medicinal plants, and a better understanding of the microbiome of T. elegans would reinforce the necessity of endophyte studies with a focus on their future applications in biotechnological areas of agriculture, medicine, and the environment.

Control of Diatraea saccharalis by the endophytic Pantoea agglomerans 33.1 expressing cry1Ac7.

Despite the fact that Bacillus thuringiensis (Bt) is found in more than 90 % of the products used against insects, it has some difficulty reaching the internal regions where the larvae feed. To solve this problem, many genetically modified microorganisms that colonize the same pests have been developed. Thus, the endophytic bacterium Pantoea agglomerans (33.1), which has been recently described as a promising sugarcane growth promoter, was genetically modified with the pJTT vector (which carries the gene cry1Ac7) to control the sugarcane borer, Diatraea saccharalis. Firstly, the bioassays for D. saccharalis control by 33.1:pJTT were conducted with an artificial diet. A new in vivo methodology was also developed, which confirmed the partial control of larvae by 33.1:pJTT. The 33.1:pJTT strain was inoculated into sugarcane stalks containing the D. saccharalis larvae. In the sugarcane stalks, 33.1:pJTT was able to increase the mortality of D. saccharalis larvae, impair larval development and decrease larval weight. Sugarcane seedlings were inoculated with 33.1:pJTT, and re-isolation confirmed the capacity of 33.1:pJTT to continuously colonize the sugarcane. These results prove that P. agglomerans (33.1), a sugarcane growth promoter, can be improved by expressing the Cry protein, and the resulting strain is able to control the sugarcane borer.

Molecular characterization by amplified ribosomal DNA restriction analysis and antimicrobial potential of endophytic fungi isolated from Luehea divaricata (Malvaceae) against plant pathogenic fungi and pathogenic bacteria.

Luehea divaricata is an important plant in popular medicine; it is used for its depurative, anti-inflammatory, and other therapeutic activities. We evaluated the antimicrobial activity of endophytic fungi isolated from leaves of L. divaricata against phytopathogens and pathogenic bacteria, and characterized the isolates based on amplified ribosomal DNA restriction analysis (ARDRA). The in vitro antagonistic activity of these endophytes against the phytopathogen Alternaria alternata was assayed by dual culture technique. Based on this evaluation of antimicrobial activity, we extracted secondary metabolites from nine endophytic fungi by partitioning in ethyl acetate and methanol. These were tested against the phytopathogens A. alternata, Colletotrichum sp and Moniliophthora perniciosa, and against the human pathogenic bacteria Escherichia coli and Staphylococcus aureus. Molecular characterization by ARDRA technique was used for phylogenetic analysis, based on comparison with sequences in GenBank. The endophytes had varied effects on A. alternata. One isolate produced an inhibition halo against M. perniciosa and against E. coli. This antibiosis activity indicates a role in the protection of the plant against microbial pathogens in nature, with potential for pharmaceutical and agricultural applications. Based on ARDRA, the 13 isolates were grouped. We found three different haplotypes of Phomopsis sp, showing interspecific variability. It appears that examination of the microbial community associated with medicinal plants of tropical regions has potential as a useful strategy to look for species with biotechnological applications.

In silico analysis of diverse endophytic fungi by using ITS1-5,8S-ITS2 sequences with isolates from various plant families in Brazil.

Brazil has a great diversity of plants, and considering that all plant species studied to date have endophytic microorganisms (bacteria or fungi), the country is a resource in the search for bioactive compounds. Endophytes live within plants without causing damage and may be in dynamic equilibrium with the health of the plant. Endophytic fungi can be identified by sequencing the region corresponding to internal transcribed spacer 1-5,8S-internal transcribed spacer 2 ribosomal DNA, and carrying out phylogenetic analyses of these sequences helps to identify species. The objective of this research was to perform in silico phylogenetic analysis of fungi isolated from various plant families in Brazil. For this study, we chose 12 articles published between 2005 and 2012 that examined endophytes isolated in Brazil. We analyzed sequences deposited in the National Center for Biotechnology Information GenBank database and carried out alignment to determine the genetic distance of strains using the Molecular Evolutionary Genetics Analysis version 5 program. The articles yielded 73 plant species belonging to 13 families found in the Brazilian States of Amazonas, Bahia, Minas Gerais, Paraná, and São Paulo. The use of GenBank and the Molecular Evolutionary Genetics Analysis program for phylogenetic observation revealed that several endophytes had been incorrectly identified because inconsistencies were apparent in their location in the phylogenetic tree. However, approximately 98% of the sequences deposited in GenBank were consistent with the identification of related genera, indicating that the database is sufficiently robust to support future studies, in which molecular identification of endophytes is made via analysis of ribosomal DNA sequences.