RESUMEN
A total of 18 paralogs of xyloglucan-specific endoglucanases (EGLs) from the glycosyl hydrolase family 12 were identified and characterized in Phytophthora sojae and Phytophthora ramorum. These genes encode predicted extracellular enzymes, with sizes ranging from 189 to 435 amino acid residues, that would be capable of hydrolyzing the xyloglucan component of the host cell wall. In two cases, four and six functional copies of these genes were found in tight succession within a region of 5 and 18 kb, respectively. The overall gene copy number and relative organization appeared well conserved between P. sojae and P. ramorum, with apparent synteny in this region of their respective genomes. Phylogenetic analyses of Phytophthora endoglucanases of family 12 and other known members of EGL 12, revealed a close relatedness with a fairly conserved gene sub-family containing, among others, sequences from the fungi Emericella desertorum and Aspergillus aculeatus. This is the first report of family 12 EGLs present in plant pathogenic eukaryotes.
Asunto(s)
Duplicación de Gen , Phytophthora/genética , Proteínas Algáceas/clasificación , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Celulasa/genética , ADN de Algas/química , ADN de Algas/genética , Expresión Génica/genética , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Phytophthora/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de AminoácidoRESUMEN
Glucose-6-phosphate isomerase (GPI) plays a key role in both glycolysis and gluconeogenesis. Isoforms of GPI are common, and therefore, its isozyme pattern is widely used to characterize isolates of Phytophthora infestans. Despite the importance of GPI in P. infestans studies, the gene encoding this enzyme has not yet been characterized. Furthermore, it has been suggested that P. infestans contains multiple copies of the gene but this hypothesis remains to be demonstrated. We have cloned and characterized GPI in various isolates of P. infestans as well as in several species of the genus Phytophthora. The gene contains 1671bp and encodes a protein with a predicted molecular weight of 60.8kDa. Multiple different alleles were identified and Southern analysis indicated certain P. infestans isolates carry several copies of the gene. Phylogenetic analysis revealed that P. infestans GPI is most closely related to sequences from Toxoplasma gondii, Arabidopsis thaliana, and Clarkia lewisii.
Asunto(s)
Glucosa-6-Fosfato Isomerasa/genética , Phytophthora/genética , Alelos , Secuencia de Bases , Cartilla de ADN , Geografía , Isoenzimas/genética , Filogenia , Phytophthora/clasificación , Phytophthora/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
Since the early 1990s, the epidemic of green mold on the cultivated mushroom Agaricus bisporus in North America has been caused by Trichoderma aggressivum f. aggressivum. The findings of earlier research suggested that the microevolutionary emergence of T. aggressivum f. aggressivum coincided with the onset of the epidemic. This hypothesis was tested further by determining the disease susceptibility of mushroom strains grown widely before the epidemic manifested. The results of complementary methods of analysis, which entailed a grain protection assay and cropping trials, established that two pre-epidemic strains were more susceptible to green mold than three post-epidemic strains being cultivated at the time of the epidemic. Thus, if T. aggressivum f. aggressivum had been present within cultivated mushrooms prior to the epidemic, it should have been detected. It still appears to be true that T. aggressivum f. aggressivum emerged during the 1990s in a manner that remains unclear.
RESUMEN
Two complementary DNA (cDNA) libraries were constructed from tissues isolated from primordia and basidiomes of Agaricus bisporus to characterize genes involved in mushroom development. Using single-pass sequencing of 869 cDNA clones, we found 477 expressed sequence tags (ESTs), including 466 not previously described in the databases for A. bisporus. A BLASTX search revealed that 374 ESTs had similarities with protein sequences available from databases; 193 of these ESTs were categorized according to their putative function. Most ESTs were assigned to one of four roles: metabolism (23%), cell structure (15%), cell growth and division (12%), and protein destination and storage (10%). The remaining ESTs with putative homologues were classified in 10 additional categories. Many ESTs could not be functionally assigned. Based on redundancy levels, at least 4 ESTs were preferentially expressed in each tissue type. Sequence analysis also suggested the presence of paralog tyrosinase genes in the A. bisporus genome.
Asunto(s)
Agaricus/genética , Agaricus/fisiología , Regulación del Desarrollo de la Expresión Génica , Genoma Fúngico , Agaricus/crecimiento & desarrollo , Secuencia de Aminoácidos , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Datos de Secuencia Molecular , Monofenol Monooxigenasa/genética , Plásmidos , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Estadística como AsuntoRESUMEN
We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of Trichoderma associated with green mold on the cultivated mushroom Agaricus bisporus. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s. Trichoderma harzianum biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to T. harzianum biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and Trichoderma atroviride (formerly T. harzianum biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and T. atroviride from Europe. Biotype 4 was not represented by the preepidemic isolates of Trichoderma as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.
Asunto(s)
Agaricus , Técnica del ADN Polimorfo Amplificado Aleatorio , Trichoderma/genética , Trichoderma/aislamiento & purificación , ADN de Hongos/análisis , ADN de Hongos/genética , Variación Genética , Genotipo , Trichoderma/clasificación , Trichoderma/patogenicidadRESUMEN
ABSTRACT A polymerase chain reaction-amplified DNA containing the internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 regions of the nuclear ribosomal DNA transcriptional unit was sequenced for 81 isolates of Trichoderma spp. associated with mushroom culture or used for biological control of plant pathogens. Phylogenetic analyses revealed that the biocontrol isolates were more closely related to an isolate of T. harzianum biotype 1 (Th1) than to the aggressive biotypes 2 and 4. Th1 has been isolated from mushroom compost but is not the cause of widespread green mold epidemics that have occurred during the last 12 years in Europe and North America. Three isolates of T. harzianum obtained from shiitake (Lentinula edodes; Shi1B and S3-96) and maitake (Grifola frondosa; Mai1) substrates were placed within the biocontrol group. We also found evidence suggesting that some isolates of T. harzianum originally identified as Th4 from Pennsylvania are more closely related to Th2 from Europe. Finally, considering the wide range in sequence distribution of our samples, we propose that the consensus sequence found in this investigation be used as the reference sequence for further studies involving the identification and taxonomy of T. harzianum.