Bioprospecting of <Emphasis Type="Italic">Diaporthe terebinthifolii</Emphasis> LGMF907 for antimicrobial compounds

Antibiotic-resistant bacteria have been observed with increasing frequency over the past decades, driving the search for new drugs and stimulating the interest in natural products sources. Endophytic fungi from medicinal plants represent a great source of novel bioactive compounds useful to pharmaceutical and agronomical purposes. Diaporthe terebinthifolii is an endophytic species isolated from Schinus terebinthifolius, a plant used in popular medicine for several health problems. The strain D. terebinthifolii LGMF907 was previously reported by our group to produce secondary metabolites with biological activity against phytopathogens. Based on these data, strain LGMF907 was chosen for bioprospecting against microorganisms of clinical importance and for characterization of major secondary metabolites. In this study, different culture conditions were evaluated and the biological activity of this strain was expanded. The crude extracts demonstrated high antibacterial activity against Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant S. aureus. The compounds diaporthin and orthosporin were characterized and also showed activity against the clinical microorganisms evaluated. This study discloses the first isolation of diaporthin and orthosporin from D. terebinthifolii, and revealed the potential of this endophytic fungus to produce secondary metabolites with antimicrobial activity.     

A prime inference on genetic diversity (RAPDs) in the marine fish Atherinella brasiliensis (Teleostei,Atherinopsidae) from Southern Brazil

Da Silva Cortinhas, M. C., Glienke, C., Prioli, A. J., Noleto, R. B., Matoso, D. A. and Cestari, M. M. 2010. A prime inference on genetic diversity (RAPDs) in the marine fish Atherinella brasiliensis (Teleostei, Atherinopsidae) from Southern Brazil. —Acta Zoologica (Stockholm) 91 : 242–248 As a result of the importance of Atherinella brasiliensis in estuarine environments, random amplified polymorphic DNA (RAPD) markers were used to verify the genetic diversity in A. brasiliensis from two different places in Paranaguá Bay (Paraná State) and one from the Conceição Lagoon (Santa Catarina State). Cytogenetic data have shown a high karyotypic diversity in some populations, although in others this peculiarity demonstrates rearrangements such as heterochromatinization. In the present study, a low level of genetic structuring between the samples from Conceição Lagoon compared with the others was observed through principal coordinate analysis (PCO), analysis of molecular variance and Mantel test according to 79 RAPD markers. As this specie does not perform horizontal migration and the individuals of Conceição Lagoon are isolated, three hypotheses are proposed to explain the results: (i) similar environments may show homogeneous populations not depending on the geographical distance, (ii) because vicariant events that formed the bays occurred in a recent period, the fragmentation effects over the structuring of the genetic diversity may still be low and not totally detectable by the RAPD technique and (iii) the isolation time or the number of generations may not be enough to promote a possible differentiation and genetic structuring between the specimens of these three places. The specimens of these places present a low level of differentiation and genetic structuring so we can consider them as a unique homogeneous population.     

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.     

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.     

Pustulan and branched β-galactofuranan from the phytopathogenic fungus Guignardia citricarpa, excreted from media containing glucose and sucrose

Guignardia citricarpa is a phytopathogenic fungus and the causal agent of citrus black spot. Incubation in a semi-defined media resulted in formation of exopolysaccharides [EPS(s)]. A medium containing glucose gave rise to a (1→6)-linked β-glucan (200 kD), pustulan, which was characterized by NMR and methylation analysis. A sucrose-containing medium provided a homogalactan (376 kD) and methylation analysis showed nonreducing end- (20%), 6-O- (53%) and 5,6-di-O-substituted Galf units (27%). An HMQC spectrum of the homogalactan showed C-1/H-1 signals at δ 108.2/4.820, 108.3/4.820 and 107.1/5.079, corresponding to three types of β- -Galf units. A DEPT analysis showed inverted signals (CH₂) at δ 67.8 and 67.2, corresponding to 6-O-substituted β- -Galf units, whereas a C-5 signal at δ 77.0 suggests 5-O-substitution, confirming a novel structure for a β-galactofuranan.     

Diversity of a soybean rhizobial population adapted to a Cerrados soil

One hundred isolates were trapped by soybean (Glycine max) plants inoculated with a soil from the Cerrados, the main producing area in Brazil. The soil was originally void of rhizobia able to nodulate soybean, and 15 years before received inoculant containing Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019; the area has been annually cropped with soybean since then, but with no further inoculation for the past 7 years. Enormous diversity was observed among the isolates, with thirteen serologically distinct groups, twelve protein and seven lipopolysaccharide profiles; no more than five isolates shared similar characteristics. An unexpected feature was that 48% of the isolates showed multiple reactions with the antisera to the serogroups established in the soils. Also 40% of the isolates reacted with the antiserum to B. japonicum strain SEMIA 566, that has never been introduced into the soil, probably due to dispersion from other cropping areas, associated with its high saprophytic competence; 13% of the isolates did not react with any of the antisera. Nodulation and N₂ fixation capacity also varied considerably among the isolates. Although one third of the isolates were fast growers with an acid reaction in vitro, and many formed pseudo-nodules on common bean (Phaseolus vulgaris), they shared several properties with the Bradyrhizobium inoculant strains. A high level of genetic diversity was confirmed when the DNAs were amplified with BOX and RPO1 primers, and several isolates were positioned in far different clusters in the analysis of interspersed repetitive or nif-directed sequences. Moreover, serological properties showed higher correlation with BOX than with RPO1 products. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Cerrados, and may play an important role as a biological buffer, avoiding the dominance of a particular strain.     

Engineering d‐limonene synthase down‐regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus‐growing areas worldwide. The pathogen may infect the fruit for 20–24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d ‐Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d ‐limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d ‐limonene content, an over‐accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up‐regulation of different pathogenesis‐related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.     

Horizontal transfer and hypovirulence associated with double-stranded RNA in Beauveria bassiana

Beauveria bassiana strains from different hosts and geographic origins were assayed for the presence of double-stranded RNA (dsRNA). Two of them (15.4 %) showed extra bands, with approximately 4.0–3.5 kb and 2–0.7 kb, respectively, after electrophoretic separation of undigested nucleic acids. Virus-like particles were approximately 28–30 nm diam. The dsRNA was maintained after conidiogenesis (vertical transmission) and was transmitted horizontally by hyphal anastomosis. Strains purged of dsRNA obtained after cycloheximide treatment showed increased conidial production when compared with strains carrying dsRNA particles. Bioassays demonstrated hypovirulence associated with dsRNA. The mean mortality against the insect Euschistus heros was reduced in strains containing dsRNA when compared with the isogenic dsRNA-free ones.