Pathogenic variation in Colletotrichum gloeosporioides infecting Stylosanthes spp. in a center of diversity in Brazil

Pathogenic variation in Colletotrichum gloeosporioides infecting species of the tropical pasture legume Stylosanthes at its center of diversity was determined from 296 isolates collected from wild host population and selected germ plasm of S. capitata, S. guianensis, S. scabra, and S. macrocephala in Brazil. A putative host differential set comprising 11 accessions was selected from a bioassay of 18 isolates on 19 host accessions using principal component analysis. A similar analysis of anthracnose severity data for a subset of 195 isolates on the 11 differentials indicated that an adequate summary of pathogenic variation could be obtained using only five of these differentials. of the five differentials, S. seabrana 'Primar' was resistant and S. scabra 'Fitzroy' was susceptible to most isolates. A cluster analysis was used to determine eight natural race clusters using the 195 isolates. Linear discriminant functions were developed for eight race clusters using the 195 isolates as the training data set, and these were applied to classify a test data set of the remaining 101 isolates. All except 11 isolates of the test data set were classified into one of the eight race clusters. Over 10% of the 296 isolates were weakly pathogenic to all five differentials and another 40% were virulent on just one differential. The unclassified isolates represent six new races with unique virulence combinations, of which one isolate is virulent on all five differentials. The majority of isolates came from six field sites, and Shannon's index of diversity indicated considerable variation between sites. Pathogenic diversity was extensive at three sites where selected germ plasm were under evaluation, and complex race clusters and unclassified isolates representing new races were more prevalent at these sites compared with sites containing wild Stylosanthes populations.

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

EVALUATION OF DIFFERENT TECHNIQUES FOR THE DIFFERENTIATION OF PATHOGENIC AND NON PATHOGENIC STRAINS OF YERSINIA-ENTEROCOLITICA

Different methods and tests have been used to evaluate the pathogenic potential of distinct Y. enterocolitica serotypes and biotypes. We tested a total of 60 Y. enterocolitica strains, being 25 of human origin (serotype O3 biotype 4 and serotype O5 biotype 1); 6 of animal origin (serotype O3 biotype 4); 19 isolated from the environment (serotype O5.27 biotypes 1 and 2); and 8 isolated from food (serotype O5 biotype 1 and serotype 05.27 biotype 1). The methods used were based on plasmid gene expression (autoagglutination, calcium-dependence at 37 degrees C and Congo Red absorption tests), chromosomal gene expression (assays for pyrazinamidase activity, salicin fermentation and esculin hydrolysis), and invasion of HEp-2 cells. All but one of the Y. enterocolitica O3 strains, were found to be potentially pathogenic when submitted to the pyrazinamidase-salicin-esculin tests. In contrast, the results obtained with the assays related to plasmidial gene expression were not so uniform, probably due to plasmid loss. The least homogeneous results were obtained with the HEp-2 cell invasion test. Y. enterocolitica O5 behaved in a uniform manner when tested with the first two groups of tests (based on chromosomal and plasmidial gene expression), but not when tested with the HEp-2 invasion assay. The strains of serotype O5.27 biotype 1 presented a uniform behavior hen submitted to the chromosomic-related tests, showing no pathogenicity. However, they did not provide conclusive results with the tests related to plasmidial gene expression or HEp-2 cell invasion. We conclude that the tests related to chromosomal gene expression (esculin-salicin-pyrazinamidase) are simple and highly effective for the detection of potentially pathogenic Y. enterocolitica isolated from clinical cases.

Variability of non-mutualistic filamentous fungi associated with Atta sexdens rubropilosa nests

A survey of the filamentous fungi other than the symbiotic one found in association with Atta sexdens rubropilosa colonies was carried out. Different fungal species (27 taxa) were isolated a few days after treating the workers with toxic baits (sulfluramid; Mirex-SO), from 40 laboratory and 20 field nests. Syncephalastrum racemosum (54 %) and Escovopsis weberi (21 %), Trichoderma harzianum (38 %) and Fusarium oxysporum (23 %) were the prevalent species in laboratory and field nests, respectively. Acremonium kiliense, Acremonium strictum, E. weberi, F oxisporum, Fusarium solani, Moniliella silaveolens and T harzianum were found in both nests' groups. We revealed that many filamentous fungi can co-exist in a dormant state inside the nests of these insects and some of them appear to be tightly associated with this environment.

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf = 0.60, as a result of dye degradation. (C) 2001 Published by Elsevier B.V. B.V.

Flavonoid increase in soybean as a response to Nezara viridula injury and its effect on insect-feeding preference

The ability of the stink bug (Nezara viridula) to induce and/or increase production of chemical defenses, i.e., flavonoids, in immature seeds of five genotypes of soybean (BR-16, IAC-100, PI 227687, PI 229358, and PI 274454) was investigated under greenhouse and laboratory conditions. Samples from pods of each genotype damaged by stink bug were analyzed for flavonoid content with high performance liquid chromatography. A dual-choice test was conducted to evaluate the feeding preference of N. viridula comparing BR-16 pods treated with extracts of PI 227687 seeds (with and without stink-bug injury), with water-treated pods. Seeds of PI 227687 damaged by N. viridula presented the highest concentration (352 mug/g) of daidzin (4'-hydroxyisoflavone-7-glucoside). The same trend was observed with genistin (4',5,7-trihydroxyisoflavone-7-glucoside): PI 227687 contained 142.4 mug/g, PI 274454, 31.6 mug/g, and PI 229358, 38.9 mug/g. Seeds damaged by stink bugs had higher isoflavone contents (daidzin and genistin), compared to controls. However, after being damaged, PI 274454 and PI 229358 produced less genistin than the other genotypes and no differences in concentration between damaged and nondamaged plants of this genotypes were observed. The numbers of observations of the insect feeding and the numbers of stylet sheaths left in water-treated BR-16 pods were greater than in those treated with PI 227687 extracts. The insects fed for longer periods on BR-16 pods treated with extract of PI 227687 without injury compared to those that were treated with extract of PI 227687 previously injured by stink bugs. Extracts of PI 227687 pods ( damaged or not) were deterrent to adults of N. viridula, and insect injury increased concentrations of daidzin and genistin in PI 227687 seeds. The deterrence seemed to be more pronounced after pods had suffered stink-bug injury.

Pathogenicity of isolates of Metarhizium anisopliae (Metsch.) Sorokin towards the cattle tick Boophilus microplus (Can.) (Acari : Ixodidae) under laboratory conditions

The pathogenic activity of Metarhizium anisopliae on Boophilus microplus females was assessed using five fungal isolates. Groups of 8 engorged ticks collected from cattle on free pasture were inoculated by immersion for 5 seconds in a spore suspension containing 2.5 x 10(8) conidia/ml. The groups were transferred to moist chambers in which they were maintained at 27 +/- 1 degreesC for about 15 days, their mortality being determined every 2, or occasionally 3, days. The isolate that produced the best results in this assay was used to inoculate female ticks with spore suspensions containing 0, 7.5 x 105, 7.5 x 10(6), 7.5 x 10(7) and 7.5 x 10(8) conidia/ml, under the same conditions as described above. The pathogenic action of M. anisopliae on engorged females was clearly demonstrated. Isolates E9 and AM were more effective, causing high tick mortality as well as reduced oviposition. The concentration of 7.5 x 10(8) conidia/ml was the most effective, with the fungus sporulating on 91.1% of the ticks. Mean percent oviposition was highest in the control treatment and lowest in the treatment with 7.5 x 10(8) conidia/ml.

PATHOGENIC OR POTENTIALLY PATHOGENIC BACTERIA AS CONTAMINANTS OF FRESH-WATER FROM DIFFERENT SOURCES IN ARARAQUARA, BRAZIL

The microbiological quality of various fresh waters in the Araraquara region, state of São Paulo, Brazil was investigated. Ninety-nine water samples were taken from rivers, reservoirs, artesian and non-artesian wells, springs and tap waters, and these waters were analysed using: plate counts of heterotrophic microorganisms (per 1 ml); Most Probable Number (MPN) of fecal coliforms and E. coli (per 100 ml); tests for presence of Salmonella, Shigella, Yersinia, the E. coli pathogens of classes EPEC, ETEC and EIEC and Mycobacterium, Shigella, Yersinia and enteroinvasive E. coli (EIEC) were not isolated. The other types of microorganisms were isolated in varying proportions. We conclude that the waters investigated represent a potential microbiological health risk.

A model for optimal chemical control of leaf area damaged by fungi population - Parameter dependence

We present a model to study a fungi population submitted to chemical control, incorporating the fungicide application directly into the model. From that, we obtain an optimal control strategy that minimizes both the fungicide application (cost) and leaf area damaged by fungi population during the interval between the moment when the disease is detected (t = 0) and the time of harvest (t = t(f)). Initially, the parameters of the model are considered constant. Later, we consider the apparent infection rate depending on the time (and the temperature) and do some simulations to illustrate and to compare with the constant case.

Coffee leaf scorch caused by a strain of Xylella fastidiosa from citrus

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var, robusta 'Apuatao 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosn and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X, fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

Soilborne filamentous fungi in Brazil

The Atlantic Rainforest is a Brazilian ecosystem that is being rapidly being destroyed, along with the abiotic and biotic factors present in it. Among the biotic factors, the fungi are found in the soil which, besides being of major importance in terms of ecological niches, also have broad and significant applications in biotechnology. In order to assess the biodiversity of these microorganisms in this type of ecosystem, the Banhado Grande region was chosen at the Jureia-Itatins Ecology Station, in the state of São Paulo, Brazil. Within this region, two areas were delimited for study, one covered with natural (primary) vegetation and the other containing vegetation that regenerated following the planting of rice crops, referred to here as secondary. Collection of compound soil samples were taken (depth 0-15 cm) over a period of two and a half years, with the litter first being removed, during dry/cold and humid/hot periods. After sifting the samples, they were appropriately processed using the serial dilution technique to isolate the fungi from the soil. Six different culture media were used, having pHs of 4.5, 7.0 and 9.0. Altogether, 1,211 strains were isolated, divided into the following groups: Hyphomycetes, the most abundant followed by Ascomycetes, Zygomycetes, Coelomycetes, and Oomycetes. From these, 112 species were identified, 8 down to the genus level, and those that did not produce conidia were grouped as Mycelia sterilia. Among the strains, 67 were cellulolytic, 32 originated solely in soil under natural vegetation, and 26 originated solely in soil under secondary vegetation.