Neiva Tinti de Oliveira

Randomly Amplified Polymorphic DNA of Trichoderma isolates and antagonism against Rhizoctonia solani

Random Amplified Polymorphic DNA (RAPD) procedure was used to examine the genetic variability among fourteen isolates of Trichoderma and their ability to antagonize Rhizoctonia solani using a dual-culture assay for correlation among RAPD products and their hardness to R. solani. Seven oligodeoxynucleotide primers were selected for the RAPD assays which resulted in 197 bands for 14 isolates of Trichoderma. The data were entered into a binary matrix and a similarity matrix was constructed using DICE similarity (SD) index. A UPGMA cluster based on SD values was generated using NTSYS (Numerical Taxonomy System, Applied Biostatistics) computer program. A mean coefficient of similarity obtained for pairwise comparisons among the most antagonics isolates was around 40%. The results presented here showed that the variability among the isolates of Trichoderma was very high. No relationship was found between the polymorphism showed by the isolates and their hardness, origin and substrata.

Genetic variability within Fusarium solani specie as revealed by PCR-fingerprinting based on pcr markers

Fusarium solani fungus (teleomorph Haematonectria haematococca) is of relevance for agriculture, producing a disease that causes significant losses for many cultivars. Moreover, F. solani is an opportunistic pathogen to animals and humans. The complexity associated to its correct identification by traditional methods justifies the efforts of using molecular markers for isolates characterization. In this work, three PCR-based methods (one PCR-ribotyping and two PCR-fingerprinting) were used to investigate the molecular variability of eighteen F. solani isolates from four Brazilian States, collected from different substrates. Genetic analysis revealed the intraspecific variability within the F. solani isolates, without any correlation to their geographical origin and substrate. Its polymorphism was observed even in the very conserved sequence of rDNA locus, and the SPAR marker (GTG)5 showed the highest polymorphism. Together, those results may contribute to understand the relation between fungal genetic variability and cultivars resistance phenotypes to fungal-caused diseases, helping plant-breeding programs.

Endophytic fungi associated with transgenic and non-transgenic cotton

Transgenic Bt cotton expresses the Cry1Ac protein from Bacillus thuringiensis, which could influence the plants capability to host endophytic fungi. The diversity of endophytic fungi in leaves, stems and roots from transgenic (Bt) and its isoline (non-Bt) cotton was evaluated during different plant developmental stages to investigate possible non-target effects of genetically modified cotton on endophytic fungal communities. A total of 17 genera of endophytic fungi were isolated. The most frequently isolated species were Phomopsis archeri from leaves and stems and Phoma destructiva from roots. While the Bt modification had no effect on endophytes, the cotton tissue and the plant developmental stage significantly influenced the diversity and composition of the fungal community. These results represent the first evaluation of the composition of endophytic fungi associated with transgenic cotton plants.

DNA polymorphism and total protein in mutants of Metarhizium anisopliae var. Anisopliae (Metsch.) Sorokin strain E9

Five mutants (MaE10, MaE27, MaE24, MaE41 e MaE49) of Metarhizium anisopliae wild strain E9 were analysed for DNA profile through the RAPD technique and for changes in total protein content by spectrophotometry, polyacrylamide gel electrophoresis and densitometry. The pattern of RAPD markers showed genetic polymorphism among the strains: out of twenty primers seven were selected, producing 113 bands. Forty seven bands were present in all strains (41.6% of monomorphic bands) and 66 showed polymorphism (58.4%). The mean coefficient of similarity among all strains was 0.75 (75%). The total protein content varied, staining in the interval of 6.0-8.0 µg/µl. The electrophoresis analysis, through zymogram and protein fraction profiles by densitometry, allowed the observation of seven bands for the wild strain E9 and five bands for the mutants MaE10, MaE27, MaE34, MaE41 and MaE49, evidence of variations in µg% among protein fractions. The RAPD technique was very sensitive to detect genetic differences between the wild type and the mutants obtained through gamma radiation. The total protein analysis also showed changes in quantity and pattern of bands after electrophoresis in the mutants compared to the wild type.

Biological and chemical control of Sclerotinia sclerotiorum using Trichoderma spp. and Ulocladium atrum and pathogenicity to bean plants

Four isolates of Sclerotinia sclerotiorum were tested for pathogenicity in IPA-10 variety bean plants (Phaseolus vulgaris L.), and all were pathogenic. Biological control in vitro was evaluated using eight isolates of Trichoderma spp. and, one of Ulocladium atrum. Chemical control in vitro with fungicides Thiophanate methyl, Iprodione and Carbendazim was also tested. Except U. atrum, all Trichoderma isolates showed antagonistic potential against S. sclerotiorum, where isolate 3601 presented the best performance. Thiophanate methyl chemical control was the most efficient. This fungicide and isolate 3601were compared in vivo in greenhouse. There was statistical difference between the treatments, and the application of fungicide and antagonist before the pathogen was the most efficient approach, reducing the percentage of pathogenicity to 32.94% and 37.04%, respectively.

Phylogenetic analysis of Monascus and new species from honey, pollen and nests of stingless bees

The genus Monascus was described by van Tieghem (1884) to accommodate M. ruber and M. mucoroides, two species with non-ostiolate ascomata. Species delimitation in the genus is still mainly based on phenotypic characters, and taxonomic studies that include sequence data are limited. The genus is of economic importance. Species are used in fermented Asian foods as food colourants (e.g. ‘red rice’ (ang-kak, angka)) and found as spoilage organisms, and recently Monascus was found to be essential in the lifecycle of stingless bees. In this study, a polyphasic approach was applied combining morphological characters, ITS, LSU, β-tubulin, calmodulin and RNA polymerase II second largest subunit sequences and extrolite data, to delimit species and to study phylogenetic relationships in Monascus. Furthermore, 30 Monascus isolates from honey, pollen and nests of stingless bees in Brazil were included. Based on this polyphasic approach, the genus Monascus is resolved in nine species, including three new species associated with stingless bees (M. flavipigmentosus sp. nov., M. mellicola sp. nov., M. recifensis sp. nov., M. argentinensis, M. floridanus, M. lunisporas, M. pallens, M. purpureus, M. ruber), and split in two new sections (section Floridani sect. nov., section Rubri sect. nov.). Phylogenetic analysis showed that the xerophile Monascus eremophilus does not belong in Monascus and monophyly in Monascus is restored with the transfer of M. eremophilus to Penicillium (P. eremophilum comb. nov.). A list of accepted and excluded Monascus and Basipetospora species is given, together with information on (ex-)types cultures and barcode sequence data.

Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects

Microbial control of insects is based on the rational use of pathogens to maintain environmentally balanced pest population levels, and Metarhizium anisopliae has been the most studied and most utilized fungal species for that purpose. The natural genetic variability of entomopathogenic fungi is considered one of the principal advantages of microbial insect control. The inter- and intraspecific variability and the genetic diversity and population structures of Metarhizium and other entomopathogenic fungi have been examined using ITS-RFLP, ISSR, and ISSP molecular markers. The persistence of M. anisopliae in the soil and its possible effects on the structures of resident microbial communities must be considered when selecting isolates for biological insect control.

Cellulase activity of a Lentinula edodes (Berk.) Pegl. strain grown in media containing Carboximetilcellulose or microcrystalline cellulose

Neste trabalho foram estudadas as atividades de endoglucanase, exocelobiohidrolase e celobiase em uma linhagem de Lentinula edodes (Berk.) Pegl. cultivada em meio liquido contendo carboximetilcelulose (CMC) ou celulose microcristalina (Avicel). Foram detectadas as atividades de endoglucanase e exocelobiohidrolase no sobrenadante das culturas crescidas tanto em meios contendo CMC como nos meios contendo Avicel, sendo observada a influencia da concentracao e do tipo de celulose. Nao foi detectada atividade de celobiase nos sobrenadantes, sendo a mesma detectada somente no extrato micelial. Com uma concentracao de 1,7% de Avicel, a linhagem estudada nao demonstrou atividade de exocelobiohidrolase. Porem, a concentracao de 0,5% obteve-se uma atividade de 74 µmol de RBBR/mg de proteina/min. Com a substituicao de Avicel por CMC a 0,5%, a atividade de exocelobiohidrolase foi reduzida a menos de 50%. A maxima atividade de endoglucanase em sobrenadantes obtidos em meio com Avicel a 0,5% foi em torno de 800 UI/mg de proteina, apos 96 horas de cultivo. Em sobrenadantes obtidos de meio com CMC, a atividade desta enzima foi de apenas 200 UI/mg de proteina.

Hyperparasitism of Cladosporium uredinicola over Puccinia puta on the host Ipomoea fistulosa

Under natural conditions a fungus, identified as Cladosporium uredinicola , was observed living as a parasite over rust lesions produced by Puccinia puta on leaves of Ipomoea fistulosa , a shrub commonly found in the State of Pernambuco, northeast of Brazil. Although recorded as a parasite of different rust species, C. uredinicola had never been mentioned as a hyperparasite of Puccinia puta . This information might be important for studies on the biocontrol of rust species.

Fungemia by Candida pelliculosa ( Pichia anomala ) in a Neonatal Intensive Care Unit: A Possible Clonal Origin

Neonatal candidemia can occur, however, infections caused by Candida pelliculosa are rare. Here, we describe an outbreak of candidemia caused by C. pelliculosa among babies hospitalized in a neonatal intensive care unit.