R. de Leon

Polymorphisms in the β-tubulin gene of Cryptosporidium parvum differentiate between isolates based on animal host but not geographic origin

Polymerase chain reaction primers were designed to target a region of the Cryptosporidium parvum beta-tubulin gene spanning an intron. Amplification products contained 11 polymorphic positions, representing a sequence divergence of 1.8%, which discriminated between isolates of C. parvum found solely in humans (genotype 1) and those found in humans and animals (genotype 2). Seven of the polymorphic sites were located outside of the intron and the polymorphism between isolates was readily demonstrated by HaeIII restriction digestion. However, all of the sequences from genotype 1 human-derived oocysts isolated in the United States and Australia were conserved. Also, there were no sequence differences between bovine isolates obtained from both continents. Therefore, isolates could not be differentiated based on geographic source of origin.

Growth of mushrooms on wheat straw and coffee pulp: Strain selection

Abstract The growth of twenty-four strains of white rot fungi on wheat straw and on mixtures of wheat straw and pressed coffee pulp was studied. Fruiting bodies were collected from some of the assays, with protein contents ranging from 15% to 38%, dry basis. The Pleurotus species showed the best results, in terms of mycelial growth, production of fruiting bodies and digestibility of residual materials, when straw was the substrate and also adequate results when mixtures of straw-pulp were used. Weight losses of the straw used for the growth of these strains ranged from 28% to 47% approximately. Lignin degradation ranged from 24% to 55%, cellulose degradation from 34% to 42%. The straw was also the best substrate in terms of relative digestibility of the residual material after cultivation of Pleurotus (75% to 138% of original value).

White-rot fungal growth on sugarcane lignocellulosic residue

SummaryTwelve white-rot fungi were grown in solid state culture on sugarcane chips previously fermented by yeast employing the EX-FERM process. The lignocellulosic sugarcane residue had 12.5% permanganate lignin and 81.3% holocellulose. After 5 to 6 weeks at 20° C, all fungi produced a solid residue which had a lower in vitro dry matter enzymatic digestibility than the original bagasse, with the exception of Coriolus versicolor which showed a slight increase of 0.6 units. Four fungi produced a residue with higher soluble solids than the original sample. Lignin losses were rather similar for all fungi tested, an average value of 38.64% of the original value was obtained. About the same amount of hemicellulose was degreaded, 32.22%. Most fungi showed a preference for hemicellulose hydrolysis over cellulose degradation. The two fungi that showed greater cellulolytic activity were Sporotrichum pulverulentum and Dichomitus squalens. No appreciable dry matter losses were detected for Agrocybe aergerita and Flammulina velutipes.

Biological pretreatment of coffee pulp

Abstract The presence of several antiphysiological factors limit the use of coffee pulp in monogastric and ruminant feeding. Among these the characteristics of its lignocellulosic fraction is important. Twenty-six white rot fungi were grown under solid substrate conditions, using previously ensiled and pressed coffee pulp without adding additional sources of nitrogen; the objective was to quantify the changes of the lignocellulosic polymer matrix and the enzymatic digestibilities. All grew and wholly covered the surface of the substrate. Six of the fungi produced fruiting bodies, which were easy to see since basidiomycetes produce large fuiting bodies. The weight loss interval ranged between 6·7 and 28·0% dry matter before fructification and from 17·0 to 48·7% after fructification. Trametes versicolor was a very fast grower and showed the highest rates of holocellulose and lignin degradation. Hemicellulose was selectively preferred over cellulose by most fungi and at least two degraded more than 80% of the original hemicellulose. Six white rots produced a solid residue with less lignin content than the one found in the original substrate. Two of them, Sporotrichum pulverulentum and Stropharia rugosoannulata , also increased the water-soluble solids and produced a solid residue with the same enzymatic digestibility as the one found for the original coffee pulp.

Intra-isolate Heterogeneity and Reproducibility of PCR-based Genotyping of Cryptosporidium parvum Using the β-tubulin Gene

Cryptosporidium parvum is a common contaminant in surface waters and presents significant problems for the water industry, public health and agriculture. Consequently, ascertaining the contaminating source of waterborne oocysts is of paramount importance. Based on currently available information, isolates of C. parvum can be differentiated into at least two genotypes using polymorphic genetic markers: genotype 1, to date isolated almost exclusively from humans, and genotype 2 isolates from humans and many other animals. Differentiation into these two genotypes has been based on either restriction fragment length polymorphisms or sequencing of PCR amplified gene fragments. The objective of this study was to evaluate the reproducibility of genotyping methods using a single isolate of C. parvum. A 620 bp fragment of the C. parvum β-tubulin gene, generated by PCR from multiple aliquots of a single preparation of oocysts of the Iowa isolate, was sequenced. Significant sequence heterogeneity was detected within this single isolate; there was more sequence variation between clones originating from the Iowa isolate (up to 0.9 %) than between individual clones originating from different isolates of C. parvum. Over 6 % of the β-tubulin gene sequence positions (38 out of 620 bp) were variable when comparing multiple clones from the one isolate. The results indicated that while the various procedures used for genotyping isolates may introduce some sequence errors, the Iowa isolate used for this investigation appeared to be composed of multiple sub-genotypes. While none of the sequence variations resulted in clones of the Iowa isolate (genotype 2) being mis-identified as genotype 1, the results have important implications if minor sequence variations are to be used for subtyping isolates and drawing conclusions regarding the origin of, or relationships between, C. parvum oocysts in water and the community.

Effect of dilution rate on the biological nitrogen fixation in thePhaseolusvulgaris-Rhizobiumphaseoli symbiosis

SummaryContinuous culture was used to produceRhizobiumphaseoli cells at different dilution rates.Phaseolusvulgaris seeds in sterile Leonard jars were then inoculated with the cells produced, and the system was left to interact for six weeks. Dry weight of the plants and number and weight of the nodules were measured. These data were compared to the response obtained with cells produced in batch culture. An increase in plant biomass and nodule weight and number were observed at higher dilution rates.

Storage in water ofRhizobiumphaseoli cells harvested from continuous culture and its effect on biological nitrogen fixation

SummaryTaken cells from cultures ofRhizobiumphaseoli grown at different dilution rates were tested for plant growth promotion in a bioassay withPhaseolusvulgaris, before and after storage in water for one month. Cells grown at higher dilution rates showed a greater loss of effectiveness