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Los gases efecto invernadero contribuyen de manera directa al cambio climático, actualmente uno de los principales problemas de la humanidad, existiendo en años recientes una preocupación debida al incremento en sus emisiones como resultado de la actividad humana en general y de la ganadería en particular. El propósito de esta nota es difundir algunos aspectos de este inconveniente proponiendo una alternativa para tratar de disminuir este problema. Dentro de los sistemas de producción pecuaria los rumiantes son los principales implicados ya que emiten alrededor de 115 millones de toneladas de metano por año, correspondiendo aproximadamente al 20% de las emisiones mundiales. La metanogénesis representa una pérdida de energía para el rumiante en términos de nutrición; y en términos ambientales contribuye al incremento de los gases con efecto invernadero por lo que el número de investigaciones a fin de reducirla se ha incrementado. Es importante dar a conocer la preopcupación de los investigadores e

Summary The plant pathogenic fungus Fusarium fujikuroi is the causal agent of bakanae disease on rice due to its ability to produce gibberellins. Besides these phytohormones, F. fujikuroi is able to produce several other secondary metabolites (SMs). Although much progress has been made in the field of secondary metabolism, the transcriptional regulation of SM biosynthesis is complex and still incompletely understood. Environmental conditions, global as well as pathway-specific regulators and chromatin remodelling have been shown to play major roles. Here, the role of FfSge1, a homologue of the morphological switch regulators Wor1 and Ryp1 in Candida albicans and Histoplasma capsulatum, respectively, is explored with emphasis on secondary metabolism. FfSge1 is not required for formation of conidia and pathogenicity but is involved in vegetative growth. Transcriptome analysis of the mutant Δffsge1 compared with the wild type, as well as comparative chemical analysis between the wild type, Δffsge1 and OE:FfSGE1, revealed that FfSge1 functions as a global activator of secondary metabolism in F. fujikuroi. Double mutants of FfSGE1 and other SM regulatory genes brought insights into the hierarchical regulation of secondary metabolism. In addition, FfSge1 is also required for expression of a yet uncharacterized SM gene cluster containing a non-canonical non-ribosomal peptide synthetase.

The generation of knock-out mutants in fungal pathogens by gene replacement and insertional mutagenesis is the classical method to validate virulence factors. An alternative strategy consists of silencing the candidate virulence gene by making use of the phenomenon of RNA interference (RNAi), adding features such as the possibility of generating knock-down mutants with variable expression levels of the target gene or the ability to simultaneously target multiple genes. Two different approaches have been assayed to generate knock-down mutants by RNAi in the phytopathogenic fungus Botrytis cinerea. In the first one, the single nitrate reductase gene in the B. cinerea genome, niaD, was silenced by transformation with a construct containing a 400-bp niaD fragment between two opposing promoters, so that a dsRNA fragment was generated. As an alternative approach, the mgfp4 gene coding for the green fluorescent protein (GFP) was silenced by transforming two different GFP-expressing strains of B. cinerea with a hairpin RNA (hpRNA)-expressing vector, containing two inverted copies of a 300-bp mgfp4 fragment separated by a spacer DNA. While the opposing dual-promoter strategy produced gene silencing in about half of the transformants assayed, the efficiency of the hpRNA-expressing vector was higher, inducing a decrease in GFP levels in more than 90 % of transformants. The degree of silencing achieved was high with both methods, but the hpRNA strategy resulted in a higher proportion of strongly silenced transformants.

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low-molecular weight plant compounds, and we have collected the proteins secreted during the first 16 hours by a double precipitation protocol. 2D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by liquid chromatography-tandem mass spectrometry of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.

Current DNA extraction protocols for genomic DNA from Botrytis cinerea almost always start with mycelium that has been reduced to powder with liquid N(2) in a mortar, and this makes their application to a large number of samples slow and cumbersome. Here we present an adaptation of an existing method [Möller et al. (1992) Nucleic Acids Res 20: 6115-6116] for which the initial steps have been modified, including the homogenization of the fungus with sand and the aid of a common household drill. This method allows the processing of large number of samples in much shorter times and generates an average of 4 mug DNA per sample, of sufficient quality for use in PCR and Southern blotting.

SUMMARY Genetic transformation is generally carried out in Botrytis cinerea by random integration of the foreign DNA into the genome, resulting in transformants that show differences among them in, for example, the expression of a reporter gene. Here we report a system for site-directed integration in which a novel recipient strain containing a 5′-truncated copy of the hygromycin resistance gene, hph, is transformed with a vector containing another truncated copy, now in the opposite end, of the same selection marker. Homologous recombination in the region shared by these two truncated copies of hph is the only way by which antibiotic-resistant transformants can be generated. The transformation frequency obtained for the site-directed strategy was only three-fold lower than that of the standard transformation, and all the transformants had at least one copy of the plasmid integrated at the expected locus. This system was tested by the expression of the green fluorescent protein and we found that the levels of this protein were more homogeneous among the transformants, when compared with those obtained by random integration. On the other hand, in this paper, we also tried to optimize gene replacements in B. cinerea, which are generally carried out by transformation with an antibiotic resistance marker flanked by regions homologous to the target gene. We studied the influence of the length of these regions on the frequency of replacement of the B. cinerea gene cel5A. Lengths between 500 and 2000 bp gave similar frequencies (about 60%), while lengths of 100 bp decreased the frequency to 6%, showing that 500 bp is a convenient size that would give optimal gene replacement frequencies in this fungus.

The cel5A gene, coding for an extracellular endo-s-1,4-glucanase, has been cloned from the phytopathogenic fungus Botrytis cinerea. The gene contains only one 62-bp intron and encodes a putative protein of 424 amino acids with a signal peptide, a family-5 glycosyl hydrolase domain, and a serine-threonine-rich domain of unknown function. The levels of cel5A transcript and extracellular s-1,4-glucanase activity were regulated by the carbon source in the same way: both were induced by carboxymethylcellulose and repressed by glucose. The cel5A mRNA could also be detected during the infection of tomato leaves by B. cinerea. Disruption of cel5A results in a strain with identical virulence as the wild-type on tomato leaves or gerbera petals, with no significant reduction in the extracellular s-1,4-glucanase activity, and with similar growth rate with carboxymethylcellulose or Avicel as the only carbon sources.

The ascomycete plant pathogen Botrytis cinerea secretes aspartic proteinase (AP) activity. Functional analysis was carried out on five aspartic proteinase genes (Bcap1-5) reported previously. Single and double mutants lacking these five genes showed neither a reduced secreted proteolytic activity, nor a reduction in virulence and they showed no alteration in sensitivity to antifungal proteins purified from grape juice. Scrutiny of the B. cinerea genome revealed the presence of nine additional Bcap genes, denoted Bcap6-14. The product of the Bcap8 gene was found to constitute up to 23% of the total protein secreted by B. cinerea. Bcap8-deficient mutants secreted approximately 70% less AP activity but were just as virulent as the wild-type strain. Phylogenetic analysis showed that Bcap8 has orthologs in many basidiomycetes but only few ascomycetes including the biocontrol fungus Trichoderma harzanium. Potential functions of the 14 APs in B. cinerea are discussed based on their sequence characteristics, phylogeny and predicted localization. Fil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina. Wageningen University. Laboratory of Phytopathology; Países Bajos Fil: Espino, José J.. Universidad de la Laguna; España Fil: Dekkers, Ester. Wageningen University. Laboratory of Phytopathology; Países Bajos Fil: Van Sluyter, Steven C.. The Australian Wine Research Institute; Australia. The University Of Melbourne; Australia Fil: Brito, Nélida. Universidad de la Laguna; España Fil: Kay, John. Cardiff University; Reino Unido Fil: González, Celedonio. Universidad de la Laguna; España Fil: van Kan, Jan A. L.. Wageningen University. Laboratory of Phytopathology; Países Bajos