Manuel Montero

Expression of an α‐1,3‐glucanase during mycoparasitic interaction of Trichoderma asperellum

Trichoderma species have been investigated as biological control agents for over 70 years owing to their ability to antagonize plant pathogenic fungi. Mycoparasitism, one of the main mechanisms involved in the antagonistic activity of Trichoderma strains, depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. The antifungal activity of an α‐1,3‐glucanase (EC 3.2.1.59, enzymes able to degrade α‐1,3‐glucans and also named mutanases) has been described in T. harzianum and its role in mycoparasitic processes has been suggested. In this study, we report on the purification, characterization and cloning of an exo‐α‐1,3‐glucanase, namely AGN13.2, from the antagonistic fungus T. asperellum T32. Expression at the transcription level in confrontation assays against the strawberry pathogen Botrytis cinerea strongly supports the role of AGN13.2 during the antagonistic action of T. asperellum.

Cell wall-degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA taxonomic species

Trichoderma is known for being the most frequently used biocontrol agent in agriculture. A fundamental part of the Trichoderma antifungal system relies on a series of genes coding for a variety of extracellular lytic enzymes. Characterization of the polymorphism between five putative isoenzymatic activities [β-1,3-glucanase (EC 3.2.1.39, EC 3.2.1.58), β-1,6-glucanase (EC 3.2.1.75), cellulase (EC 3.2.1.4; EC 3.2.1.21, EC 3.2.1.91), chitinase (EC 3.2.1.30, EC 3.2.1.52), protease (EC 3.4.11; EC 3.4.13–19; EC 3.4.21–24, EC 3.4.99)] was carried out using 18 strains from three sections of Trichoderma. Of these, seven strains were from T. sect. Pachybasium, nine from T. sect. Trichoderma and two from T. sect. Longibrachiatum. Thirty-seven different alleles in total were identified: 13 for β-1,3-glucanase, four for β-1,6-glucanase, three for cellulase, eight for chitinase and nine for protease activity. A dendrogram (constructed by the unweighted pair group method with arithmetic averages) based on isoenzymatic data separated the 18 strains into three main enzymatic groups: T. harzianum, T. atroviride/T. viride/T. koningii and T. asperellum/T. hamatum/T. longibrachiatum. Isoenzymatic groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location, based on their sequence of internal transcribed spacer 1 in ribosomal DNA and their antifungal activities.

BGN16.3, a novel acidic β-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

A new component of the β‐1,6‐glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo‐β‐1,6‐glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic β‐1,6‐glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to β‐1,6‐glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with β‐1,6‐glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement β‐1,6‐glucanases in this process is discussed.

Cloning and characterization of bgn16·3, coding for a β‐1,6‐glucanase expressed during Trichoderma harzianum mycoparasitism

Aims:  To clone and characterize the gene coding for BGN16·3, a β‐1,6‐glucanase putatively implicated in mycoparasitism by Trichoderma harzianum, a biocontrol agent used against plant pathogenic fungi.

Morphological and functional study of the GH-immunoreactive adenohypophyseal cells in ovariectomized rats

SummaryIn view of the existence of a different secretion pattern of growth hormone (GH) between male and female rats, the aim of the present study was to analyse the role played by ovarian steroid hormones in the modulation of such secretion. To do so, postpuberal female rats were ovariectomized and killed at 30 days after the operation. The basal serum levels of growth hormone, together with cell area, cytoplasmic area and nuclear area of the hypophyseal somatotropic cells of normal and ovariectomized rats were compared. The results obtained show that ovariectomy induces a significant decrease (p<0.05) in the basal serum levels of GH, accompanied by an increase in cellular and cytoplasmic areas, with no significant differences in nuclear area. Overiectomy was also accompanied by an increase in reaction intensity and the number of GH-immunoreactive cells (p<0.01). These findings point to the shift towards a masculine secretory and morphological pattern following ovariectomy and supports the hypothesis that ovarian steroids intervene in the establishment of a different pattern in females compared to males.

Estrogenic modulation of met-enkephalin-induced prolactin secretion in rats.

SummaryIn order to elucidate the possible role of estrogens in the sex-dependent modulation of met-enkephalin-induced prolactin secretion, and the cellular response at adenohypophyseal level, analyses of serum prolactin levels together with a morphometric study of prolactin-immunoreactive cells were carried out following intraventricular administration of the opioid to estrogenprimed male rats. Administration of the opioid led to an increase in the serum levels of the hormone and, morphometrically, a significant increase in the cellular, cytoplasmic and nuclear areas was observed. When naloxone was administered previously, the above, mentioned changes did not appear, and the values found were similar to those observed in the animals treated only with estrogens. The results suggest that the met-enkephalin-induced prolactin secretion is modulated by the gonadal steroid environment.

In vivo and in vitro evidence of growth hormone-releasing factor-like produced locally in the adenohypophyseal cells of the rat

In order to characterize immunocytochemically the existence of GRF in the rat adenohypophysis and the origin of this hormone, an immunocytochemical and morphometric study was made of r-GRF-immunoreactive cells from the adenohypophysis of untreated adult rats, adult rats treated intraventricularly with colchicine and in primary cultures of adult rat adenohypophyseal cells that had been incubated with serum devoid of GRF. r-GRF immunoreactive cells were observed in untreated rat pituitaries, both male and female, although the numbers of positive cells were greater in the males (p less than 0.05) and were found to increase in number following treatment with colchicine (p less than 0.01). These cells appeared dispersed throughout the anterior lobe, without forming clusters, and were often close to blood vessels. Additionally, immunoreactive cells appeared in the cultures at 7 days of incubation. The presence of GRF-immunoreactive cells in the adenohypophysis of rats previously treated with colchicine suggests the existence of a non-hypothalamic origin for r-GRF; this is confirmed by the findings obtained in the in vitro studies which would corroborate the hypothesis that the origin of the neuropeptide is in the rat adenohypophysis itself.

Morphometric study of immunoreactive GH-cells of the adenohypophysis of rats treated with Met-enkephalin

Morphological and morphometric changes were found in adenohypophyseal GH immunoreactive cells after intraventricular administration of Met-enkephalin to adult rats of both sexes. Morphologically, following Met-enkephalin treatment, the males showed cells stained intensely and homogeneously in their cytoplasm, while the females showed cells with a weaker degree of staining and a cytoplasm with a granular aspect; previous administration of naloxone prevented the changes observed in the males and intensified the reaction in the females. Morphometrically, in all the groups of animals studied, the males showed a greater cellular area than the females, accompanied by a decrease in the cytoplasmic area but without significant differences in the nuclear area. Met-enkephalin in both sexes produced an increase in cellular area, accompanied by a significant increase in the cytoplasmic area and, although less manifest, in the nuclear area. Neither kind of change appeared when naloxone was administered prior to Met-enkephalin treatment.

Morphometric analysis of GH-immunoreactive cells after GH release induced by treatment with estrogen in adult male rats

In order to elucidate the response of somatotropic cells to the influence of gonadal steroids on the regulation of growth hormone secretory patterns, a morphometric analysis was carried out on the GH-immunoreactive cells of adult male rats treated chronically with intramuscular injections of estradiol valerate. The morphometric and morphological results obtained were correlated to the serum levels of GH at the moment of sacrifice. Treatment with a daily dose of 125 micrograms of estradiol vaterate over 15 d was seen to lead to an increase (p less than 0.01) in the serum GH values accompanied by a decrease in the intensity of reaction of the GH-cells and, morphometrically, a reduction in their size (p less than 0.01) due to a decrease in the cytoplasmic area (p less than 0.01), but without significant changes in the nuclear area. Our results suggest that in male rats estrogens enhance the release of the intracellular GH pool but that they do not affect hormone synthesis to a great extent.