Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Utility of six fungicides for selective isolation of Evlachovaea sp. and Tolypocladium cylindrosporum.

The effects of the fungicides dodine, benomyl, thiabendazole, mancozeb, cupric sulfate, and copper oxychloride were examined in vitro upon germination and further development of Evlachovaea sp. and Tolypocladium cylindrosporum. Fungicidal activity depended on concentrations and varied among products, fungi and the strains tested. Depending on the fungicidal concentration, germination of conidia was induced but germlings produced neither mycelium nor new conidia. There was a good recovery of both Evlachovaea sp. and T. cylindrosporum from previously sterilized soils with fungicide-supplemented medium. Fungi were resistant to copper oxychloride up to 30 g/l, and this fungicide was found to have no utility for a selective medium. Minimal fungicide concentrations for successful isolations were 1 mg/l for benomyl, 200 mg/l for cupric sulfate, 50 mg/l for dodine, 100 mg/l for mancozeb, and 4 mg/l for thiabendazole. Thiabendazole, which is easy to obtain and can be used in low quantities, showed the greatest utility for a selective medium with these entomopathogenic fungi.

In vitro susceptibility to fungicides by invertebrate-pathogenic and saprobic fungi.

The effect of five fungicides, benomyl (1 mg/l), dodine (50 mg/l), manzate (100 mg/l), cupric sulphate (200 mg/l) and thiabendazole (4 mg/l) was tested under in vitro conditions on development of 15 isolates of fungi pathogenic for insects and other invertebrates (Beauveria brongniartii, Culicinomyces clavisporus, Duddingtonia flagrans, Hirsutella thompsonii, two Metarhizium anisopliae, Nomuraea rileyi, two Isaria/Paecilomyces spp., and Sporothrix insectorum) and 13 isolates of contaminant fungi (five Aspergillus spp., Cladosporium cladosporioides, Cunninghamella echinulata, Fusarium roseum, Gliocladium sp., Mortierella isabellina, Mucor plumbeus, Rhizopus arrhizus and Trichothecium roseum) originating mostly from tree-hole breeding sites of mosquitoes. Most pathogenic and contaminant fungi had clear patterns of susceptibility or resistance to tested concentration of the fungicide. Development of both pathogenic and contaminant fungi on fungicide-supplemented medium varied among fungi and fungicides tested. Minimal inhibition of pathogenic fungi was found for cupric sulphate, benomyl, dodine, thiabendazole < manzate. The highest inhibition of contaminants was obtained with thiabendazole > benomyl and dodine > manzate and cupric sulphate. Thiabendazole was the most appropriate fungicide to isolate fungi pathogenic to invertebrates from substrates with high water contents and rich in organic material. The results underline the importance of adapting both a fungicide and its concentration for a selective medium for isolating specific target fungi and while selecting against possible contaminants.

New insights into the cell cycle profile of Paracoccidioides brasiliensis.

The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus' multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus' unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature.

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae.

The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 microg/ml (agronomic dose), 10.62 microg/ml and 10 microg/ml. G. mosseae was able to germinate in the presence of 2.12 microg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 microg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi.

The nucleation of microtubules in Aspergillus nidulans germlings

Microtúbulos säo filamentos compostos por dímeros das tubulinas alfa e beta e têm uma variedade de funçöes nas células vivas. Em fungos, os corpúsculos polares dos fusos säo geralmente considerados os centros organizadores dos microtúbulos. Com o objetivo de contribuir para uma melhor compreensäo dos processos de nucleaçäo dos microtúbulos no fungo filamentoso A. nidulans, nós utilizamos a droga antimicrotúbulo Benomil em experimentos de bloqueio e liberaçäo para depolimerizar e repolimerizar os microtúbulos. Após 20 segundos de reincubaçäo em meio sem Benomil, pequenos microtúbulos foram formados a partir de pontos distribuídos pela célula, sugerindo que os pontos de nucleaçäo de microtúbulos säo aleatoriamente distribuídos pelas hifas de A. nidulans. Como em A. nidulans o movimento nuclear é dependente de microtúbulos foi analisado se mutantes defectivos na distribuiçäo de núcleos ao longo das hifas (mutantes nud) possuíam algum defeito evidente nos microtúbulos. Os microtúbulos citoplasmáticos, dos fusos e astrais estäo presentes e aparentam-se normais em todos os mutantes nud, mas foi observada uma pequena distorçäo na proporçäo de fusos mitóticos longos e curtos nestes mutantes, comparados com o controle. Isto sugere que alguns núcleos de mutantes nud näo alcançam a fase tardia da divisäo celular, em temperatura näo restritiva.

Transformation of the entomopathogenic fungi, paecilomyces fumosoroseus and paecilomyces lilacinus (deuteromycotina: hyphomycetes) to benomyl resistence

The entomopathogenic fungi Paecilomyces fumosoroseus and P. lilacinus have been transformed to resistance to the fungicide benomyl by a polyethylene glycol (PEG)-mediated procedure using a mutant b-tubulin gene from Neurospora crassa carried on plasmid pBT6. Benomyl-resistant transformants of P. lilacinus were obtained that could tolerate greater than 30 µg/ml benomyl and P. fumosoroseus transformants were obtained that could tolerate 20 µg/ml benomyl. Following 5 serial passages of transformants on benomyl-containing media and 5 serial passages on non-selective media, 100 per cent of P. lilacinus transformants were found to be mitotically stable by a conidial germination test. In contrast, only 4 out of 9 transformants of P. fumosoroseus were mitotically stable. Southern blot analysis of genomic DNA from both species suggested that the mechanism of transformation in all transformants was by gene replacement of the b-tubulin allele. Non-homologous vector sequences were not detectable in the genomes of transformants.

Action against Vibrio cholerae O1 Tox+ of chemical products used in the lemon production.

Tucuman is the first lemon exporting province in Argentina and the fourth lemon exporter in the world. The present work was set up to study the survival of Vibrio cholerae O1 Tox+ after application of different chemical products used in the lemon production (from its cultivation until its packing). The following products were studied: copper oxychloride, benomil (a carbamate), active chlorine, sodium-o-phenylphenoate, guazatine (a polyamine mixture), imazalil (an imidazole) and fresh and dehydrated lemon peel. Using different dilutions of the products above mentioned antimicrobial tests were carried out with different exposure times against V. cholerae Serogroup O1, Biotype El Tor, Serotype Inaba. The microorganism was used at concentrations of 10(2), 10(4), 10(6) and 10(8) CFU ml-1, the latter one being considered as an infectious dose. The following results were obtained: 1) Active chlorine (chlorinated water) showed bactericidal activity at concentrations of 0.5 x 10(-1), 10(-1), y 2 x 10(-1) g l-1 after 10 min of exposure time. 2) Copper oxychloride, sodium-o-phenylphenoate, guazatine and imazalil showed bactericidal activity against V. cholerae at concentrations of 10(2) and 10(4) CFU ml-1. 3) Due to the fact that the fruit is successively sprayed with several chemical products during its cultivation, it could be proposed that the result of the successive treatments is superior to the result of a treatment with each of the individual products. This consideration should be taken into account when evaluating the eventual protection of the lemon.

Bactericidal activity against Vibrio cholerae of chemical products used in lemon production in Tucumán, Argentina.

The present research was set up to verify whether the chemical products used in lemon production (from cultivation until packaging) have a bactericidal or a bacteriostatic ability against Vibrio cholerae O1. The studied products were: copper oxychloride, benomil (a carbamate), active chlorine, sodium-o-phenylphenoate, guazatine (a polyamine mixture), imazalil (an imidazole) and lemon peel. The latter was studied with and without treatment using the above mentioned chemicals. Different dilutions of these products were tried out with varying exposure times against the bacterium V. cholerae Serogroup O1, Biotype E1 Tor, Serotype Inaba. The concentrations of the microorganism ranged from 10(2) to 10(8) CFU ml-1, the latter one being considered an infectious dose. The following results were obtained: 1) active chlorine (chlorinated water) showed bactericidal activity at concentrations of 50, 100 and 200 ppm after 10 min of exposure time, 2) copper oxychloride, sodium-o-phenylphenoate, guazatine and imazalil showed bactericidal activity against V. cholerae at concentrations of 10(2) and 10(4) CFU ml-1, 3) due to the fact that during its cultivation the fruit is successively sprayed with several chemical products, it could be that the result of the successive treatments is superior to the result of a repeated treatment with each of the individual products. This consideration should be taken into account when evaluating the eventual protection of the lemon.

High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae.

Three different methods, (i) PEG, (ii) electroporation and (iii) biolistic, were employed to transform Metarhizium anisopliae using benomyl resistance as a selectable marker. Transformation frequencies and mitotic stability varied for each method, from 0.8 to 6.9 transformants micrograms-1 of DNA and 46%, respectively, by the PEG method; 1.3 to 1.8 transformants micrograms-1 of DNA and 67% by electroporation; and 32 to 201 transformants micrograms-1 of DNA and 90% by biolistic. We demonstrate by PCR that 60% of the transformants were generated by gene conversion.

Effects of selected synthetic compounds on growth of Mucor rouxii.

The in vitro activity of four synthetic compounds was tested on fungal cells from Mucor rouxii. The compounds included phenylenediamine, two phenolamines, and quinone. At the concentrations tested (10(-2)-10(-4)M) the compounds exhibited antifungal activity, with the exception of quinone. On the basis of their effects on spore germination, and development of hyphae, phenylenediamine was the more active. The mechanism of action of the compounds is still unknown, but hyphae show morphological alterations and disturb the distribution of calcofluor in the cell wall. This suggests variations in the genesis of the cell wall.

At least three genes are responsible for benomyl-resistance in Metarhizium anisopliae.

Four benomyl-resistant mutants isolated from Metarhizium anisopliae were 10 to 500 times more resistant than the original MT strain. The resistance markers analysed in three of these mutants were due to three different mutations and no additive effect of these genes was observed in double mutants. The four mutants presented normal conidiation in the presence or absence of benomyl and no sensitivity or resistance to temperature. Probably M. anisopliae has a mechanism of benomyl resistance differing from those of Aspergillus nidulans and Neurospora crassa.

Estudo da influência de agrotóxicos sobre o desenvolvimento do Rhizobium leguminosarum biovar phaseoli / Study of the agrotoxics influence over the development of the Rhizobium leguminosarum biovar phaseoli

As bactérias fixadoras de nitrogênio atmosférico, via processo simbiótico, repassam N2 às leguminosas sob forma de amônia, proporcionando aumento na produçäo de alimentos protéicos, reciclagem biológica de nitrogênio do ar, evitando o alto custo da adubaçäo nitrogenada e o efeito potencialmente poluidor do nitrato lixiviado. Testou-se quatro estirpes de Rhizobium phaseoli frente a quatro fungicidas indicados para o tratamento de sementes de Phaseolus vulgaris L. (feijoeiro), no Estado do Rio Grande o sul (Brasil). Considerando-se as dosagens recomendadas dos fungicidas, houve crescimento bacteriano das quatro estirpes frente a Benomil e inibiçäo total frente a Captan; porém, para PCNB e Thiram, a resistência dependeu do tipo de estirpe. Observou-se que näo há influência de fungicidas sobre o desenvolvimento do Rhizobium phaseoli quando seleciona-se a estirpe adequada ao fungicida corretamente dosado.

[Inhibition of Mucor rouxii growth by synthetic substances]. / Inhibición del crecimiento de Mucor rouxii por substancias sintéticas.

Mucor rouxii cells were used to examine the possible antimycotic activities of four substances: phenolamines, phenylendiamine and quinone. These substances are original structures recently synthesized. Assays in plates showed that 10(-2) M of phenolamines and phenylendiamines give rise to halos of growth inhibition. Assays in liquid media using 10(-4) M of substances showed 100% inhibition of spore germination. Specifically, the phenylendiamine showed 49% inhibition on development of mycelium. In these cells the calcofluor distribution changes, suggesting alterations in cell wall. No inhibition of growth was found using the quinone. The activity for substances were evaluated using standard antifungal benomyl. On this basis, the substance phenylendiamine it is an antimycotic active. The mechanism of action is not presently known.