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.     

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.     

Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini)

Endophyte microorganisms are organisms that live inside plants without causing any apparent damage to their hosts. Since all plants exhibit endophyte microorganisms, it is believed that mutual association is of great importance in nature. Luehea divaricata (Martius & Zuccarini), known popularly in Brazil as agoita-cavalo, is a big-sized tree with a wide distribution in the country that possesses medicinal qualities for: dysentery, leucorrhea, rheumatism, blennorrhoea, tumors, bronchitis, and depuration. This research aims at isolating and molecularly characterizing fungi isolates from L. divaricata by sequence analysis of ITS1-5.8S-ITS2 rDNA. Further, the colonization of endophyte in the host plant by Light and Scanning Electron Microscopy will also be investigated. Whereas, genera Alternaria, Cochliobolus, Diaporthe, Epicoccum, Guignardia, Phoma, and Phomopsis, were identified; rDNA sequence analysis revealed intra-species variability among endophyte isolates of the genus Phomopsis sp. Light and Scanning Electron Microscopy techniques showed the presence of endophyte fungi inside L. divaricata leaves, inhabiting inter- and intra-cellular spaces. These types of extensive colonization and dissemination were reported throughout all the leaf parts in palisade parenchyma, esclerenchyma, spongy parenchyma, adaxial epidermis, and vascular bundle indicating colonization of endophytes in múltiple structural sub-niches in the host plant.     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

Embryo abortion and histological features in the interspecific cross between <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. and <Emphasis Type="Italic">P. coccineus</Emphasis> L

In this paper, the causes of early embryo abortion in the reciprocal crosses between Phaseolus vulgaris L. (a cultivar) and Phaseolus coccineus L. (a wild form) were studied. Methacrylate resin sections, 3–5 μm thick, of 3 to 14 day-old seeds were used to examine the embryo developmental stages and the state of seed tissue. It was observed that, embryos aborted at different developmental stages (globular to early cotyledon) depending on the maternal parent. The use of P. coccineus cytoplasm resulted in a higher number of abortion than in reciprocal crosses. Many of them took place between 5 and 6 days after pollination (DAP). Histological analyses permitted to observe that the embryo development was slower in the cross between P. coccineus and P. vulgaris, compared to parental seeds. It would be related to a deficient endosperm development in reciprocal crosses and, in some extent, hypertrophy of the suspensor might be the main cause of early embryo abortion. Then, it would be practical to overcome this incompatibility by rescuing the embryo at the globular stage of development.     

Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity

The compound 3-nitropropionic acid is a potent neurotoxic agent in animals and well-known as a potent inhibitor of Mycobacterium tuberculosis. In this research, we were able to extract this compound from the endophytic fungus, Phomopsis longicolla (FJ62759), isolated from Trichilia elegans A. JUSS ssp. elegans. The aim of this study was the isolation of secondary metabolites produced by P. longicolla, the chemical identification of these compounds and evaluation of their antimicrobial and insecticidal activity. To accomplish these goals, the fungus was cultured in BD broth for 25 days without agitation at 28 °C, and then the broth was separated from the mycelium. The supernatant was partitioned with dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), and butanol (BuOH) solvents resulting in 3 extracts. However, only the EtOAc extract was used for fractionation and chemical identification because it had the greatest mass. After common chromatographic procedures, the fractions were analyzed by nuclear magnetic resonance to elucidate the chemical components. This procedure resulted in the identification of 3-nitropropionic acid in the D fraction. Evaluation of the insecticidal and antimicrobial activity of this compound has been accomplished, and the results indicate good inhibition of the citrus pathogen Guignardia citricarpa and cocoa pathogen Moniliophthora perniciosa and slight inhibition of the human bacterial pathogens Micrococcus luteus, Salmonella typhi and slight inhibition of phytopathogenic bacteria Xanthomonas axonopodis pv. phaseoli. The evaluation of insecticide activity did not show mortality of the Diatraea saccharalis larvae by the metabolite 3-nitropropionic acid in the D fraction. The results suggest that P. longicolla is a bioactive metabolic producing endophytic fungus with biotechnological properties.     

Endophytic fungi: expanding the arsenal of industrial enzyme producers

Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of the aerial plant parts, particularly in leaf sheaths, sometimes even within the bark and root system without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce a wide range of enzymes and secondary metabolites exhibiting a variety of biological activities. However, they have been only barely exploited as sources of enzymes of industrial interest. This review emphasizes the suitability and possible advantages of including the endophytic fungi in the screening of new enzyme producing organisms as well as in studies aiming to optimize the production of enzymes through well-known culture processes. Apparently endophytic fungi possess the two types of extracellular enzymatic systems necessary to degrade the vegetal biomass: (1) the hydrolytic system responsible for polysaccharide degradation consisting mainly in xylanases and cellulases; and (2) the unique oxidative ligninolytic system, which degrades lignin and opens phenyl rings, comprises mainly laccases, ligninases and peroxidases. The obvious ability of endophytic fungi to degrade the complex structure of lignocellulose makes them useful in the exploration of the lignocellulosic biomass for the production of fuel ethanol and other value-added commodity chemicals. In addition to this, endophytic fungi may become new sources of industrially useful enzymes such as lipases, amylases and proteases.     

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)