16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril).

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.

Outbreak of copper poisoning in cattle fed poultry litter.

In a feedlot of about 1,000 head of cattle, 146 animals died within a period of a few months affected by a disease characterized by anorexia, icterus, hemoglobinuria, constipation, or diarrhea. The clinical course of the disease lasted a few days. Postmortem findings were generalized icterus and a yellow discolored liver. The kidneys were dark brown, and the urinary bladder was filled with urine of the same dark-brown color. The main histopathological findings were centrolobular coagulative necrosis, apoptosis, bilestasis, and proliferation of bile ducts in the portal space. Changes in the kidneys included nephrosis and the presence of bile and precipitates, and cylinders of albumin and of hemoglobin in the uriniferous tubules. Liver samples, collected from 3 animals on which postmortem examinations were performed, had 2,008, 2,783 and 4,906 ppm copper in their dry matter. Two samples of poultry litter fed to the cattle contained 362 and 323 ppm copper. The green forage that formed the rest of their feed only had 4.7 ppm copper. Copper poisoning was diagnosed, most probably caused by feeding litter from poultry that had been fed a ration treated with copper sulfate to avoid aspergillosis.

Effect of high sugar concentration on nitrogenase activity of Acetobacter diazotrophicus.

Acetobacter diazotrophicus is a nitrogen-fixing bacterium that growth inside sugar cane plant tissue where the sucrose concentration is approximately 10%. The influence of high sugar content on nitrogenase was measured in the presence of oxygen and of nitrogen added in the form of ammonium and amino acids. In all parameters analyzed, 10% sucrose protected nitrogenase against inhibition by oxygen, ammonium, some amino acids, and also to some extent by salt stress. The oxygen concentration at which inhibition occurred increased from 2 kPa in 1% glucose or gluconic acid, to 4 kPa (0.4 atm) in 10% sucrose. Nitrogenase activity was partially inhibited by increased ammonium levels (2.0, 5.0, and 10.0 mM) in the presence of 1% sucrose, but the cells maintained their nitrogenase activity at 10% sucrose. This could be explained by the slow ammonium assimilation by the cells in the presence of high sucrose concentrations, i.e., independent of its concentration between 2 and 10 mM, the assimilation of ammonium was reduced to one-third in cells grown with 10% sucrose. Some amino acids were also tested in the presence of 1 and 10% sucrose. Cells grown in 1% sucrose had their nitrogenase activity reduced by 50-98% in the presence of glutamic acid, glutamine, alanine, asparagine, or threonine, whereas with 10% sucrose, nitrogenase activity was increased by glutamic acid and was reduced by only 61-73% by the other amino acids. The effect of NaCl concentrations (0.0, 0.25, 0.5, 0.75, or 1.0%) was also studied at the two concentrations of sucrose. Nitrogenase activity and growth of A. diazotrophicus, which was visualized by the pellicle formation in semi-solid medium, showed sensitivity even to low NaCl concentrations, which was somewhat relieved at the higher sucrose level. These observations indicate different osmotolerance mechanisms for sucrose and salt.

Phylogenetic identification of two major nitrogen-fixing bacteria associated with sugarcane.

Acetobacter diazotrophicus and Herbaspirillum seropedicae were identified by genetic methods based on 16S rRNA sequences. A specific PCR method in combination with probing was developed for A. diazotrophicus. The PCR system includes four primers, of which the primers named AC (CTGTTTCCCGCAAGGGAC) and DI (GCGCCCCATTGCTGGGTT) generated an 445 bp amplicon in all of the 11 A. diazotrophicus strains tested. The phylogenetic position of H. seropedicae was determined. H. seropedicae forms with Oxalobacter formigenes a separate lineage in the beta-subclass of Proteobacteria.

Emended description of Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans, a milk plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3.

[Pseudomonas] rubrisubalbicans, a mild plant pathogen. Herbaspirillum seropedicae, and EF group 1 strains (clustered by an immunological method) were investigated by a polyphasic approach with DNA-rRNA and DNA-DNA hybridizations and auxanography on 147 substrates. Our results show that they all belong to the genus Herbaspirillum. In addition to H. seropedicae, two other species are described: Herbaspirillum rubrisubalbicans and a new unnamed species, Herbaspirillum species 3, containing mainly strains of clinical origin. The three species can be differentiated on the basis of their auxanographic features and DNA-DNA similarities. The type strain of H. rubrisubalbicans is NCPPB 1027 (=LMG 2286); representative strains of the third Herbaspirillum species are strains CCUG 189 (=LMG 5523), CCUG 10263 (=LMG 5934), and CCUG 11060 (=LMG 5321). It has been confirmed that H. rubrisubalbicans is an endophytic diazotroph. It colonizes the roots, the stems, and predominantly the leaves of sugarcane (Saccharum spp.), while Herbaspirillum seropedicae colonizes in large numbers many different species of the Gramineae. Both diazotrophic Herbaspirillum species could be differentiated with meso-erythritol and N-acetylglucosamine. Oligonucleotide probes based on partial sequences of the 23S rRNA of H. seropedicae and H. rubrisubalbicans (HS and HR probes, respectively), were constructed and used as diagnostic probes.

Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat.

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that >10(3) cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10(7)/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.

Epizootic botulism of cattle in Brazil.

The first diagnosis of botulism in cattle in Brazil and its epizootiology are reviewed. The high prevalence of the disease raised on phosphorus deficient pastures in Savanna regions has caused severe economic losses in the past. The temperature induced microcomplement fixation test (TIMCF) confirmed the clinical-pathological diagnosis in all of the 24 cases studied by this method. The most important reason why botulism has not been controlled satisfactorily in Brazil is the lack of an available effective vaccine (type C and D). Additional prophylactic measures are phosphorus supplementation and removal of carcasses from the pasture.

[The etiology of "Cara inchada," a periodontal disease of young cattle in Brazil]. / Zur Atiologie der "Cara inchada", einer parodontalen Erkrankung der Jungrinder in Brasilien.

A sequence of research on "Cara inchada", a periodontal disease of young cattle (CI), involving heavy losses in Brazil, is described and results of laboratory investigations and field experiments are reported. A suspicion of being a primary nutritional disease of the skeleton could not be confirmed. In the other hand, bacteria, especially of the genus Bacteroides, were isolated from the periodontal CI-lesions. These bacteria possess enough pathogenic potential, through the production of enzymes and endotoxins, to cause primary destruction of the periodontal tissues. The lesions of the upper jaw, and also of the mandibula, of the diseased animals were diagnosed as a purulent periodontitis and a secondary ossifying alveolar periostitis. As CI occurs enzootically on new, cultivated pastures in cleared forest and savanna areas, and as the incidence of the disease declines with the years of pasture use, in order to disappear again, it can be postulated that a determining factor exists in the soil and consequently in the pasture, the disturbance of the equilibrium of the microflora in the formerly virgin soil possibly causes a modification of the flora of the rumen and the oral cavity, so that bacteria, as Bacteroides spp., present in the subgingival space, could dominate and become pathogenic. The frequent diarrhoea observed in calves affected by CI could be a consequence of the modification of the microflora in the digestive tract. Accordingly, CI could be considered as an infectious periodontitis of calves due to altered ecological soil conditions.

Membrane protein patterns of three Azospirillum species and Herbaspirillum seropedicae.

Membrane protein patterns in SDS-PAGE clearly characterized Azospirillum amazonense, A. brasilense, A. lipoferum, and Herbaspirillum seropedicae. Each species showed also a typical major band with approximate molecular weights (AMWs) of 44, 39.5, 35.5, and 37.5 Kdaltons for the four species respectively. Characteristic lighter bands with AMWs of 63.5 and 19 Kdaltons were shown by A. amazonense. Similar bands of 54, 24 and 24.5 Kdaltons were typical of A. brazilense, A. lipoferum and H. seropedicae, respectively. The three Azospirillum species showed four identical proteins in common, out of the six main proteins presented in SDS-PAGE; H. seropedicae had only two major proteins with one of them common to all three Azospirillum species. The data strongly favors the maintenance of Azospirillum and Herbaspirillum as valid generic taxa as well as supports the validity of the three Azospirillum species.

Livestock poisoning in South America by species of the Senecio plant.

The pyrrolizidine alkaloids retrorsine, senecionine, integerrimine, neosenkirkine and florosenine were isolated from different Senecio spp. common in southern Brazil, Uruguay and Paraguay. Structure elucidation of these compounds was mainly accomplished by two-dimensional FT NMR techniques. Detailed interpretations of 1H-1H and 1H-13C NMR correlation spectra are given. The alkaloids neosenkirkine, integerrimine and florosenine were found in Senecio leptolobus De Candolle, retrorsine accompanied by senecionine in Senecio selloi (Spreng.) DC and Senecio cisplatinus Cabrera. In Senecio heterotrichus DC. integerrimine is accompanied by retrorsine. Investigations on pathology and histopathology of Senecio envenomation in Brazil are summarized.

Macrocyclic trichothecenes: cause of livestock poisoning by the Brazilian plant Baccharis coridifolia.

Nine macrocyclic trichothecenes, thought to cause toxic effects in herbivors, were isolated from the Brazilian shrub Baccharis coridifolia. The structures of two new components, named miophytocene A and B, are described. Structure analysis was mainly accomplished by application of two-dimensional Fourier transform NMR techniques. The trichothecenes are most likely produced by the soil fungus Myrothecium verrucaria, which was isolated from soil samples around B. coridifolia. It is concluded that the plant is able to absorb and accumulate these mycotoxins. Histopathological studies on rabbits which received roridin E and A per os revealed mainly necrosis and hemorrhages of the mucosa of the colon and in the case of roridin A also of the caecum. Lethality in mice was generally low, intoxication proceeding slowly. It has to be considered, however, that all trichothecenes tested are practically insoluble in aqueous solutions and were applied as emulsions (i.p. and per os application). No trichothecenes or other toxic substances were detected from the Brazilian species Baccharis dracunculifolia, although slight toxic effects have been reported in cattle.

Selective infection of maize roots by streptomycin-resistant Azospirillum lipoferum and other bacteria.

The percentage of low-level streptomycin-resistant (20 microgram/mL) bacteria in surface-sterilized or washed maize roots was more than a thousand times higher than that in soil populations. There was also a higher incidence of resistant bacteria in rhizosphere as compared with non-rhizosphere soil and bacteria isolated from maize roots were relatively tolerant to several other antibiotics. Azospirillum lipoferum was predominant in surface-sterilized roots of field-grown maize and was low-level streptomycin-resistant while most soil isolates were sensitive. Inoculation with A. brasilense isolated from wheat roots was unsuccessful in terms of establishment even when streptomycin-resistant strains were used. Unidentified causes of specific plant-bacteria affinities therefore transcend the role of antibiotic resistance in maize root infection.

A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov.

Sixty-one strains of the root-associated nitrogen fixer Spirillum lipoferum exhibited a similar morphology in peptone--succinate salts medium: vibrioid cells having a diameter of 1.0 micrometer. When grown in broth the cells had a single polar flagellum, but when grown on agar at 30 degrees C lateral flagella of shorter wavelength were also formed. The DNA base composition was 69--71 mol% guanine + cytosine when determined by thermal denaturation. DNA homology experiments indicated the occurrence of two distinct but related homology groups: 46 strains were in group I and 15 strains were in group II. Group II strains were distinguished by their ability to use glucose as a sole carbon source for growth in nitrogen-free medium, by their production of an acidic reaction in a peptone-based glucose medium, by their requirement for biotin, and by their formation of wider, longer, S-shaped or helical cells in semisolid nitrogen-free malate medium. The results indicate that two species exist, and on the basis of their characteristics it is proposed that they be assigned to a new genus, Azospirillum. Strians belonging to group II are named A. lipoferum (Beijerinck) comb. nov., while those belonging to group I are named A. brasilense sp. nov. Strain Sp 59b (ATCC29707) is proposed as the neotype strain for A. lipoferum, and strain Sp 7 (ATCC 29145) is proposed as the type strain for A. brasilense.

Nitrate and nitrite reductase negative mutants of N2-fixing Azospirillum spp.

Chlorate resistant spontaneous mutants of Azospirillum spp. (syn. Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants from A. brasilense and 13 from A. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr-). Most of the mutants were also nitrite reductase negative (nir-), only 3 remaining nir+. Two mutants from nr+ nir+ parent strains lost only nir and became like the nr+ nir- parent strain of A. brasilense. No parent strain or nr+ mutant showed any nitrogenase activity with 10 mM NO3-. In all nr- mutants, nitrogenase was unaffected by 10 mM NO3-. Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir-. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr- nir-) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.

Light microscopy observations of tetrazolium-reducing bacteria in the endorhizosphere of maize and other grasses in Brazil.

Roots of field-grown tropical maize, Panicum maximum Jacq. and Digitaria decumbens Stent., and of sorghum and wheat grown in monoxenic culture with the diazotroph Spirillum lipoferum (syn. Azospirillum spp.) were examined for tetrazolium-reducing bacteria following incubation of roots in a malate-phosphate buffer-2,3,5-triphenyltetrazolium chloride medium. Bacteria were observed between and in cells of the cortex, in intercellular spaces between the cortex and endodermis, in xylem cells, and in and between pith cells. In maize, colonization of the inner cortex and stele appears to occur in the absence of significant bacterial colonization or collapse of outerlying tissues. Bacteria in the stele remained viable after a 6-h treatment of roots with chloramine-t, indicating that the endodermis was intact. Infection of the inner cortex and stele appears to occur initially in branches, and then to spread longitudinally into main roots. Inter- and intra-cellular infections of the cortex were observed in monoxenic systems. Tetrazolium reduction and prominent crystal formation was not specific for diazotrophic bacteria, but S. lipoferum was isolated from surface-sterilized roots, and S. lipoferum-like organisms were observed in the endorhizosphere. A correlation of inner cortex and stele infections with the presence of branches appears to explain previous observations that excised roots of grasses exhibiting high nitrogenase activity are characteristically branched roots with an intact cortex.

Ultrastructural evaluation of parathyroid glands and thyroid C cells of cattle fed Solanum malacoxylon.

Fine structural alterations of thyroid C cells and parathyroid chief cells were evaluated after feeding dried leaves of the calcinogenic plant, Solanum malacoxylon, to cattle for 1, 6 and 32 days. Thyroid C cells initially were degranulated in response to the hypercalcemia, and parathyroid chief cells accumulated secretory granules. There was hypertrophy of thyroid C cells with well-developed secretory organelles but few secretory granules in the cytoplasm after 6 days of feeding S. malacoxylon. Inactive chief cells with dispersed profiles of endoplasmic reticulum and increased lysosomal bodies predominated in the parathyroid glands. Multiple foci of soft tissue mineralization were present in the heart, lung, and kidney. Thyroid C cells underwent hypertrophy and hyperplasia after 32 days of S. malacoxylon, and parathyroid chief cells were inactive or atrophic in response to the long-term hypercalcemia. Severe soft tissue mineralization was present throughout the cardiovascular system, lung, kidney, and spleen. These ultrastructural changes in thyroid C cells and parathyroid chief cells plus the widespread soft tissue mineralization observed after feeding cattle small amounts of S. malacoxylon are consistent with the recent evidence that leaves of this plant are a potent source of the active metabolite, 1,25-dihydroxycholecalciferol, of vitamin D.

Denitrification by N2-fixing Sprillum lipoferum.

Forty-nine N2-fixing strains of Spirillum lipoferum isolated from a wide range of plant roots and soils were examined for reduction of NO3-. All strains reduced NO3-to NO2-. Thirty of the strains further reduced NO2-with production of gas. Examiniation of representative strains of the putative denitrifiers showed that they produced both N2O and C2H4 in the presence of 0.1 atm of C2H2. Strains which did not reduce NO2-with production of gas produced C2H4 but ont N2O in the presence of C2H2. This is the first report of a N2-fixing bacterium able to bring about denitrification of NO3.