Diversity of maize (Zea mays L.) rhizobacteria with potential to promote plant growth.

Plant growth-limiting factors, such as low nutrient availability and weak pathogen resistance, may hinder the production of several crops. Plant growth-promoting bacteria (PGPB) used in agriculture, which stimulate plant growth and development, can serve as a potential tool to mitigate or even circumvent these limitations. The present study evaluated the feasibility of using bacteria isolated from the maize rhizosphere as PGPB for the cultivation of this crop. A total of 282 isolates were collected and clustered into 57 groups based on their genetic similarity using BOX-PCR. A representative isolate from each group was selected and identified at the genus level with 16S rRNA sequencing. The identified genera included Bacillus (61.5% of the isolates), Lysinibacillus (30.52%), Pseudomonas (3.15%), Stenotrophomonas (2.91%), Paenibacillus (1.22%), Enterobacter (0.25%), Rhizobium (0.25%), and Atlantibacter (0.25%). Eleven isolates with the highest performance were selected for analyzing the possible pathways underlying plant growth promotion using biochemical and molecular techniques. Of the selected isolates, 90.9% were positive for indolepyruvate/phenylpyruvate decarboxylase, 54.4% for pyrroloquinoline quinine synthase, 36.4% for nitrogenase reductase, and 27.3% for nitrite reductase. Based on biochemical characterization, 9.1% isolates could fix nitrogen, 36.6% could solubilize phosphate, 54.5% could produce siderophores, and 90.9% could produce indole acetic acid. Enzymatic profiling revealed that the isolates could degrade starch (90.1%), cellulose (72.7%), pectin (81.8%), protein (90.9%), chitin (18.2%), urea (54.5%), and esters (45.4%). Based on the data obtained, we identified three Bacillus spp. (LGMB12, LGMB273, and LGMB426), one Stenotrophomonas sp. (LGMB417), and one Pseudomonas sp. (LGMB456) with the potential to serve as PGPB for maize. Further research is warranted to evaluate the biotechnological potential of these isolates as biofertilizers under field conditions.

Neopestalotiopsis species presenting wide dye destaining activity: report of a mycelium-associated laccase.

Wastewaters from textile dyeing industries represent an ecological concern, notably due to the known toxicity of azo dyes to the local microbiome and human health. Although physicochemical approaches are the rule for the treatment of industrial effluents, biological strategies such as enzyme-mediated dye destaining is a promising alternative. Notwithstanding a broad range of microorganisms, including fungi, algae, yeast, and bacteria, display dye-destaining properties, most of the literature has focused in ligninolytic fungi, leaving other classes of organisms somehow ignored. In this study, six endophytic strains isolated from Maytenus ilicifolia were studied for their destaining activity. The phylogenetic and morphological analysis allowed the identification of strain LGMF1504 as Neopestalotiopsis sp. LGMF1504 that decolorized several commercial dyes as the result of a mycelium-associated laccase. The enzyme expression was modulated by carbon and nitrogen content in the culture medium, it was weakly affected by the presence of aromatic compounds and metal ions while some common laccase mediators improved the destaining activity onto dye substrates. The best culture condition observed for laccase activity was a basic culture medium containing 5 g L-1 starch and 15 g L-1 ammonium tartrate. The laccase activity showed low substrate specificity and almost unaltered performance in a wide range of pH values and NaCl concentrations, suggesting the potential of Neopestalotiopsis sp. LGMF1504 for biodegradation approaches.

ERG11 gene polymorphisms and susceptibility to fluconazole in Candida isolates from diabetic and kidney transplant patients.

INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 µg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.

Muscodor brasiliensis sp. nov. produces volatile organic compounds with activity against Penicillium digitatum.

Endophytic fungi belonging to Muscodor genus are considered as promising alternatives to be used in biological control due to the production of volatile organic compounds (VOCs). The strains LGMF1255 and LGMF1256 were isolated from the medicinal plant Schinus terebinthifolius and, by morphological data and phylogenetic analysis, identified as belonging to Muscodor genus. Phylogenetic analysis suggests that strain LGMF1256 is a new species, which is herein introduced as Muscodor brasiliensis sp. nov. The analysis of VOCs production revealed that compounds phenylethyl alcohol, α-curcumene, and E (ß) farnesene until now has been reported only from M. brasiliensis, data that supports the classification of strain LGMF1256 as a new species. M. brasiliensis completely inhibited the phytopathogen P. digitatum in vitro. We also evaluated the ability of VOCs from LGMF1256 to inhibit the development of green mold symptoms by inoculation of P. digitatum in detached oranges. M. brasiliensis reduced the severity of diseases in 77%, and showed potential to be used for fruits storage and transportation to prevent the green mold symptoms development, eventually reducing the use of fungicides.

ERG11 gene polymorphisms and susceptibility to fluconazole in candida isolates from diabetic and kidney transplant patients

Abstract INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.

Differential colonization by bioprospected rhizobial bacteria associated with common bean in different cropping systems.

In this study, we evaluated the diversity of rhizobia isolated from root nodules on common bean (Phaseolus vulgaris) derived from Andean and Mesoamerican centers and grown under field and greenhouse conditions. Genetic characterization of isolates was performed by sequencing analyses of the 16S rRNA gene and 2 housekeeping genes, recA and glnII, and by the amplification of nifH. Symbiotic efficiency was evaluated by examining nodulation, plant biomass production, and plant nitrogen (N) accumulation. The influence of the environment was observed in nodulation capacity, where Rhizobium miluonense was dominant under greenhouse conditions and the Rhizobium acidisoli group prevailed under field conditions. However, strain LGMB41 fit into a separate group from the type strain of R. acidisoli in terms of multilocus phylogeny, implying that it could belong to a new species. Rhizobium miluonense LGMB73 showed the best symbiotic efficiency performance, i.e., with the highest shoot-N content (77.7 mg/plant), superior to the commercial standard strain (56.9 mg/plant). Biodiversity- and bioprospecting-associated studies are important to better understand ecosystems and to develop more effective strategies to improve plant growth using a N-fixation process.

Composition of endophytic fungal community associated with leaves of maize cultivated in south Brazilian field.

The objective of this study was to conduct a survey about fungi associated with leaves from two different maize plant lineages and to analyze their microbiota diversity. Isolated fungi were identified by morphological analysis and molecular taxonomy was performed using ITS1-5.8S-ITS2 rDNA. About 27 fungi morphotypes were obtained, 15 of them were from the first maize lineage. About 86.7% of the individuals belonged to the Dothideomycetes class (Phoma sorghina, Epicocum nigrum, Cladosporium sp., Bipolaris zeicola, and Alternaria alternata complex) and 13.3% to the Sordariomycetes class (Diaporthe/Phomopsis sp. and Nigrospora sp.). This ratio was opposite in the other maize lineage with 25.0% of Dothideomycetes (E. nigrum and Pleosporales) and 75.0% of Sordariomycetes (Gibberella fujikuroi complex, Fusarium graminearum complex, Diaporthe/Phomopsis sp., and Nigrospora sp.). By concerning the analyses of morphological characteristics and molecular phylogeny, this study intended to identify the groups of saprophytic, phytopathogenic, and mycotoxin fungi, which differently co-inhabit leaf tissue of maize plants in both tested lineages.

Identification of genes associated with asexual reproduction in Phyllosticta citricarpa mutants obtained through Agrobacterium tumefaciens transformation.

Phyllosticta citricarpa is the epidemiological agent of Citrus Black Spot (CBS) disease, which is responsible for large economic losses worldwide. CBS is characterized by the presence of spores (pycnidiospores) in dark lesions of fruit, which are also responsible for short distance dispersal of the disease. The identification of genes involved in asexual reproduction of P. citricarpa can be an alternative for directional disease control. We analyzed a library of mutants obtained through Agrobacterium tumefaciens transformation system, looking for alterations in growth and reproductive structure formation. Two mutant strains were found to have lost the ability to form pycnidia. The flanking T-DNA insertion regions were identified on P. citricarpa genome by using blast analysis and further gene prediction. The predicted genes containing the T-DNA insertions were identified as Spindle Poison Sensitivity Scp3, Ion Transport protein, and Cullin Binding proteins. The Ion Transport and Cullin Binding proteins are known to be correlated with sexual and asexual reproduction in fungi; however, the exact mechanism by which these proteins act on spore formation in P. citricarpa needs to be better characterized. The Scp3 proteins are suggested here for the first time as being associated with asexual reproduction in fungus. This protein is associated with microtubule formation, and as microtubules play an essential role as spindle machinery for chromosome segregation and cytokinesis, insertions in this gene can lead to abnormal formations, such as that observed here in P. citricarpa. We suggest these genes as new targets for fungicide development and CBS disease control, by iRNA.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients.

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Diaporthe endophytica and D. terebinthifolii from medicinal plants for biological control of Phyllosticta citricarpa.

The citrus industry is severely affected by citrus black spot (CBS), a disease caused by the pathogen Phyllosticta citricarpa. This disease causes loss of production, decrease in the market price of the fruit, and reduction in its export to the European Union. Currently, CBS disease is being treated in orchards with various pesticides and fungicides every year. One alternative to CBS disease control without harming the environment is the use of microorganisms for biological control. Diaporthe endophytica and D. terebinthifolii, isolated from the medicinal plants Maytenus ilicifolia and Schinus terebinthifolius have an inhibitory effect against P. citricarpa in vitro and in detached fruits. Moreover, D. endophytica and D. terebinthifolii were transformed by Agrobacterium tumefaciens for in vivo studies. The transformants retained the ability to control of phytopathogenic fungus P. citricarpa after transformation process. Furthermore, D. endophytica and D. terebinthifolii were able to infect and colonize citrus plants, which is confirmed by reisolation of transformants from inoculated and uninoculated leaves. Light microscopic analysis showed fungus mycelium colonizing intercellular region and oil glands of citrus, suggesting that these two new species are capable of colonizing citrus plants, in addition to controlling the pathogen P. citricarpa.

Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis.

BACKGROUND: Strain CPAC 7 (=SEMIA 5080) was recently reclassified into the new species Bradyrhizobium diazoefficiens; due to its outstanding efficiency in fixing nitrogen, it has been used in commercial inoculants for application to crops of soybean [Glycine max (L.) Merr.] in Brazil and other South American countries. Although the efficiency of B. diazoefficiens inoculant strains is well recognized, few data on their protein expression are available. RESULTS: We provided a two-dimensional proteomic reference map of CPAC 7 obtained under free-living conditions, with the successful identification of 115 spots, representing 95 different proteins. The results highlighted the expression of molecular determinants potentially related to symbiosis establishment (e.g. inositol monophosphatase, IMPase), fixation of atmospheric nitrogen (N2) (e.g. NifH) and defenses against stresses (e.g. chaperones). By using bioinformatic tools, it was possible to attribute probable functions to ten hypothetical proteins. For another ten proteins classified as "NO related COG" group, we analyzed by RT-qPCR the relative expression of their coding-genes in response to the nodulation-gene inducer genistein. Six of these genes were up-regulated, including blr0227, which may be related to polyhydroxybutyrate (PHB) biosynthesis and competitiveness for nodulation. CONCLUSIONS: The proteomic map contributed to the identification of several proteins of B. diazoefficiens under free-living conditions and our approach-combining bioinformatics and gene-expression assays-resulted in new information about unknown genes that might play important roles in the establishment of the symbiosis with soybean.

Identification and characterization of endophytic bacteria from corn (Zea mays L.) roots with biotechnological potential in agriculture.

Six endophytic bacteria of corn roots were identified as Bacillus sp. and as Enterobacter sp, by sequencing of the 16S rRNA gene. Four of the strains, CNPSo 2476, CNPSo 2477, CNPSo 2478 and CNPSo 2480 were positive for the nitrogen fixation ability evaluated through the acetylene reduction assay and amplification of nifH gene. Two Bacillus strains (CNPSo 2477 and CNPSo 2478) showed outstanding skills for the production of IAA, siderophores and lytic enzymes, but were not good candidates as growth promoters, because they reduced seed germination. However, the same strains were antagonists against the pathogenic fungi Fusarium verticillioides, Colletotrichum graminicola, Bipolaris maydis and Cercospora zea-maydis. As an indication of favorable bacterial action, Enterobacter sp. CNPSo 2480 and Bacillus sp. CNPSo 2481 increased the root volume by 44% and 39%, respectively, and the seed germination by 47% and 56%, respectively. Therefore, these two strains are good candidates for future testing as biological inoculants for corn.

Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes.

Maize is one of the most important crops worldwide, and in Brazil, the state of Paraná stands as its largest producer. The crop demands high inputs of N fertilizers, therefore all strategies aiming to optimize the grain production with lower inputs are very relevant. Endophytic bacteria have a high potential to increment maize grain yield by means of input via biological nitrogen fixation and/or plant growth promotion, in this last case increasing the absorption of water and nutrients by the plants. In this study, we established a collection of 217 endophytic bacteria, isolated from roots of four lineages and three hybrid genotypes of maize, and isolated in four different N-free culture media. Biochemical-comprising growth in different carbon sources, intrinsic tolerance to antibiotics, and biochemical tests for catalase, nitrate reductase, urease, and growth in N-free media in vitro-and genetic characterization by BOX-PCR revealed great variability among the isolates. Both commercial hybrids and homozygous lineages were broadly colonized by endophytes, and sequencing of the 16S rRNA gene revealed the presence of bacteria belonging to the genera Pantoea, Bacillus, Burkholderia, and Klebsiella. Qualitative differences in endophytic colonization were detected between lineages and hybrid genotypes.

Two-dimensional proteome reference map of Rhizobium tropici PRF 81 reveals several symbiotic determinants and strong resemblance with agrobacteria.

Rhizobium tropici strain PRF 81 is used in commercial inoculants for common-bean crops in Brazil because of its high efficiency in nitrogen fixation and, as in other strains belonging to this species, its tolerance of environmental stresses, representing a useful biological alternative to chemical nitrogen fertilizers. In this study, a proteomic reference map of PRF 81 was obtained by two-dimensional gel electrophoresis and MALDI-TOF/TOF-TOF mass spectrometry. In total, 115 spots representing 109 different proteins were successfully identified, contributing to a better understanding of the rhizobia-legume symbiosis and supporting, at proteomics level, a strong resemblance with agrobacteria.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species.

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia ("espinheira-santa") in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Col1 and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

Bioprospecting highly diverse endophytic Pestalotiopsis spp. with antibacterial properties from Maytenus ilicifolia, a medicinal plant from Brazil.

Thirteen endophytic fungal strains of the genus Pestalotiopsis were isolated from the medicinal plant Maytenus ilicifolia Mart. ex. Reiss (commonly known as "espinheira santa") and their antimicrobial properties were investigated. Two isolates were successful in inhibiting the growth of the tested microorganisms (Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA)) using the technique of bioautographic thin-layer chromatography (TLC) agar overlay assay. An analysis based on a polyphasic approach integrating taxonomic information, morphological traits, RAPD markers, and the sequencing of the ITS1-5.8S-ITS2 of the rDNA led to the assignment of the isolates as belonging to the species Pestalotiopsis microspora, Pestalotiopsis vismiae, and Pestalotiopsis leucothoes. Therefore, the present study presents a new approach to the study of endophytic fungi of the genus Pestalotiopsis.