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.

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.

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.

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 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.

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.

Cytotoxic and mutagenic effects of iodine-131 and radioprotection of acerola (Malpighia glabra L.) and beta-carotene in vitro.

The radioisotope iodine-131 [(131)I] can damage DNA. One way to prevent this is to increase the amount of antioxidants via dietary consumption. The goal of this study was to evaluate the radioprotective effect of fresh acerola pulp and synthetic beta-carotene in Rattus norvegicus hepatoma cells (HTC) in response to [(131)I] exposure in vitro. Cellular DNA damage was subsequently assessed using a cytokinesis block micronucleus assay. The mutagenic and cytotoxic activities of doses of [(131)I] (0.1, 0.5, 1, 5, and 10 µCi), acerola (0.025, 0.125, and 0.25 g acerola pulp/mL), and beta-carotene (0.2, 1, and 2 µM) were evaluated. Radioprotective tests were performed by simultaneous treatment with acerola (0.25 g/mL) plus [(131)I] (10 µCi) and beta-carotene (0.2 µM) plus [(131)I] (10 µCi). Acerola, beta-carotene, and low concentrations of [(131)I] did not induce micronucleus formation in HTC cells; in contrast, high concentrations of [(131)I] (10 µCi) were mutagenic and induced DNA damage. Moreover, neither acerola nor beta-carotene treatment was cytotoxic. However, acerola reduced the percentage of [(131)I]-induced damage, although beta-carotene did not show a similar effect. Thus, our results suggest that acerola diet supplementation may benefit patients who are exposed to [(131)I] during thyroid diagnostics and therapy.

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.

Diversity of endophytic fungal community associated with Piper hispidum (Piperaceae) leaves.

Tropical and subtropical plants are rich in endophytic community diversity. Endophytes, mainly fungi and bacteria, inhabit the healthy plant tissues without causing any damage to the hosts. These fungi can be useful for biological control of pathogens and plant growth promotion. Some plants of the genus Piper are hosts of endophytic microorganisms; however, there is little information about endophytes on Piper hispidum, a medicinal shrub used as an insecticide, astringent, diuretic, stimulant, liver treatment, and for stopping hemorrhages. We isolated the fungal endophyte community associated with P. hispidum leaves from plants in a Brazilian forest remnant. The endophytic diversity was examined based on sequencing of the ITS1-5.8S-ITS2 region of rDNA. A high colonization frequency was obtained, as expected for tropical angiosperms. Isolated endophytes were divided into 66 morphogroups, demonstrating considerable diversity. We identified 21 isolates, belonging to 11 genera (Alternaria, Bipolaris, Colletotrichum, Glomerella, Guignardia, Lasiodiplodia, Marasmius, Phlebia, Phoma, Phomopsis, and Schizophyllum); one isolate was identified only to the order level (Diaporthales). Bipolaris was the most frequent genus among the identified endophytes. Phylogenetic analysis confirmed the molecular identification of some isolates to genus level while for others it was confirmed at the species level.

Transposition of autonomous and engineered impala transposons in Fusarium oxysporum and a related species.

The impala transposon of Fusarium oxysporum is an active element. We demonstrated that the imp160 copy, transposed into the gene encoding nitrate reductase, is an autonomous element, since it excises from this gene and reinserts at a new genomic position in backgrounds free of active elements. An element in which the transposase gene was replaced by a hygromycin B resistance gene was used (1) to demonstrate the absence of endogenous transposase in several F. oxysporum strains and (2) to check the ability of different genomic copies of impala to transactivate this defective element. This two-component system allowed the identification of autonomous elements in two impala subfamilies and revealed that transactivation can occur between highly divergent elements. We also demonstrate that the autonomous copy transposes in a closely related species complex, F. moniliforme, in a fashion similar to that observed in F. oxysporium. The ability of impala to function as a two-component system and to transpose in a heterologous host promises further advances in our understanding of the factors that modulate transposition efficiency and demonstrates the potential of impala as a means of establishing a transposon tagging system for a wide range of fungal species.