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.

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.

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.

Molecular characterization of endophytes isolated from Saccharum spp based on esterase and ribosomal DNA (ITS1-5.8S-ITS2) analyses.

This study used esterases and ribosomal DNA (rDNA) markers to determine endophytic variability in order to better understand endophyte-host interactions. Polyacrylamide gel electrophoresis and esterase isoenzymes (EST; EC 3.1.1.3), with α-naphthyl acetate and ß-naphthyl acetate as substrates, were used to assess relationships among endophytes. ITS1-5.8S-ITS2 sequencing data were used as rDNA markers. Thirty-two esterases were obtained from 37 isolates of Saccharum spp, which clustered into five endophyte groups. Esterase EST-06 was observed with the highest frequency, being present in 22 of the 37 isolates analyzed, followed by esterase EST-11, which was present in 20 isolates. The esterases EST-10 and EST-14 were present in 19 isolates and EST-09 was present in 18 isolates. The esterase EST-01 was unique to isolate 33 and can, therefore, be used as a marker for this isolate. None of the esterases identified were common to all isolates tested. Similarly, phylogenetic analysis, based on rDNA sequence data, classified the isolates into 5 genus groups: 1) Curvularia with a 100% bootstrap value (BP), 2) Alternaria with 100% BP, 3) Epicoccum with 60% BP, 4) Phoma with 89% BP, and 5) Saccharicola with 100% BP. This polyphyletic analysis based on several markers, therefore, proved to be a valuable approach in determining the relationship between variation in endophytes and their associated host plants. Furthermore, both the esterase and rDNA analyses obtained similar results and were equally effective in resolving relationships.

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.

Phylogenetic diversity of endophytic leaf fungus isolates from the medicinal tree Trichilia elegans (Meliaceae).

Various types of organisms, mainly fungi and bacteria, live within vegetal organs and tissues, without causing damage to the plant. These microorganisms, which are called endophytes, can be useful for biological control and plant growth promotion; bioactive compounds from these organisms may have medical and pharmaceutical applications. Trichilia elegans (Meliaceae) is a native tree that grows abundantly in several regions of Brazil. Preparations using the leaves, seeds, bark, and roots of many species of the Meliaceae family have been widely used in traditional medicine, and some members of the Trichilia genus are used in Brazilian popular medicine. We assessed the diversity of endophytic fungi from two wild specimens of T. elegans, collected from a forest remnant, by sequencing ITS1-5.8S-ITS2 of rDNA of the isolates. The fungi were isolated and purified; 97 endophytic fungi were found; they were separated into 17 morpho-groups. Of the 97 endophytic fungi, four genera (Phomopsis, Diaporthe, Dothideomycete, and Cordyceps) with 11 morpho-groups were identified. Phomopsis was the most frequent genus among the identified endophytes. Phylogenetic analysis showed two major clades: Sordariomycetes, which includes three genera, Phomopsis, Diaporthe, and Cordyceps, and the clade Dothideomycetes, which was represented by the order Pleosporales.

Relationship between age of commercial broiler chickens and response to photostimulation.

A study was conducted to determine the relationship between age of commercial broiler chickens and response to photostimulation. The chickens were brooded collectively for 1 wk and then separated into five light treatment groups with each group replicated three times using a completely randomized block design. After Week 1, the five light treatments applied were: 6, 5, 4, 3, and 2 wk of continuous supplementary lighting, respectively. Chickens were fed a corn-soybean meal basal diet containing 22% CP and 3,300 kcal ME/ kg of feed from day old to 7 wk. At 7 wk of age, chickens in Group 5 treated with supplementary light during the last 2 wk of growth had the highest mean BW when the data on sexes were combined. When the data were separated by sex and analyzed, only male chickens showed a significant response to photostimulation and this was observed at 4 wk of age. Chickens in Group 5 had the best feed conversion with less mortality and no leg disorders. There were no significant differences among the relative organ weights among the treatment groups except for the pancreas. The pancreas taken from Group 5 had the smallest weight relative to BW. There were no significant differences in breast, thigh, drumstick, and wing weights when expressed as relative weight. However, there were significant differences when expressed as absolute weight. The breasts taken from birds in Group 5 were significantly heavier than the control breasts.