RESUMEN
Pathogens can actively alter fruit pH around the infection site, signaling modulation of pathogenicity-factor expression, as found for alkalinizing (Colletotrichum and Alternaria spp.) and acidifying (Penicillium, Botrytis, and Sclerotinia spp.) fungi. The nitrogen-metabolism genes GDH2, GS1, GLT, and MEP genes are differentially expressed during colonization by Colletotrichum gloeosporioides, and a Δgdh2 strain reduces ammonia accumulation and pathogenicity. We analyzed the contribution of transporters GLT and MEPB to C. gloeosporiodes pathogenicity. Germinating spores of Δglt strains showed reduced appressorium formation; those of ΔmepB mutants showed rapid ammonia uptake and accumulation inside the hyphae, indicating deregulated uptake. Both mutants reduced pathogenicity, indicating that these transporters function during alkalinizing species pathogenicity. We compared the expressions of these genes in C. gloeosporioides and Sclerotinia sclerotiorum, and found five to 10-fold higher expression at the transcript level in the former. Interestingly, GLT and MEPB in the alkalinizing species showed no and very low sequence identity, respectively, with their counterparts in the acidifying species. Knockout analysis of GLT and MEPB and their differential transcript regulation in the alkalinizing and acidifying species suggest that the ammonia accumulation contributing to pathogenicity in the former is modulated by factors at the gene-regulation levels that are lacking in the acidifying species.
Asunto(s)
Ascomicetos/genética , Ascomicetos/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Nitrógeno/metabolismo , Secuencia de Aminoácidos , Amoníaco/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , FilogeniaRESUMEN
The rhizobia-legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.
Asunto(s)
Celulasa/metabolismo , Fabaceae/microbiología , Raíces de Plantas/microbiología , Rhizobium leguminosarum/enzimología , Simbiosis , Celulasa/genética , Celulasa/aislamiento & purificación , Celulosa/biosíntesis , Clonación Molecular , Fabaceae/citología , Genes Bacterianos , Ligamiento Genético , Medicago/citología , Medicago/microbiología , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Raíces de Plantas/citología , Rhizobium leguminosarum/citología , Rhizobium leguminosarum/genética , Nódulos de las Raíces de las Plantas/citología , Nódulos de las Raíces de las Plantas/microbiología , Plantones/microbiologíaRESUMEN
The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.
Asunto(s)
Trichoderma/genética , Marcadores Genéticos , Variación Genética , Genotipo , Control Biológico de Vectores , Polimorfismo Genético , Técnica del ADN Polimorfo Amplificado Aleatorio , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Trichoderma/clasificaciónRESUMEN
A complete chitin synthase gene and one chitin synthase gene fragment of the zygomycete Mucor circinelloides have been cloned and analyzed. Both genes encode zymogenic Class II chitin synthases. Hybridization analysis showed that there must exist at least another Class II chitin synthase gene in M. circinelloides highly homologous to the cloned Mcchs1 and Mcchs2. The expression of these genes during the dimorphic growing stages was analyzed. Northern hybridizations showed that Mcchs1 transcript accumulates only during the exponentially growing hyphal stage, while no expression could be detected in the yeast form. Expression of Mcchs2 could not be detected at any stage. Accumulation of Mcchs1 transcript was not influenced by visible light. The existence of a multigene chitin synthase family and the observation that Mcchs1 transcription depends upon the dimorphic stage indicate that various chitin synthase activities may have different roles in the dimorphic growth of M. circinelloides.
Asunto(s)
Quitina Sintasa/genética , Mucor/genética , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Isoenzimas/genética , Datos de Secuencia Molecular , Mucor/enzimología , Mutación , Filogenia , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADNRESUMEN
The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.
Asunto(s)
Control Biológico de Vectores , Filogenia , Trichoderma/clasificación , Trichoderma/genética , ADN Mitocondrial/genética , Variación Genética , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Mapeo Restrictivo , Esporas Fúngicas , Trichoderma/aislamiento & purificaciónRESUMEN
Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.
Asunto(s)
Fusarium/genética , Fusarium/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN/genética , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Fabaceae/microbiología , Fusarium/patogenicidad , Variación Genética , Enfermedades de las Plantas/microbiología , Plantas Medicinales , Polimorfismo de Longitud del Fragmento de Restricción , EspañaRESUMEN
Electrophoretic karyotypes of ten strains of Mucor circinelloides f. lusitanicus were generated by contour-clamped homogeneous electric field (CHEF) gel-electrophoresis. Most of the strains analyzed showed polymorphisms, but a different main karyotype pattern could be correlated with each mating type. Genome structure was further analyzed by gene assignment to the chromosome-sized DNAs. Nonradioactive hybridization techniques identified the chromosomal localization of seven cloned genes. The hybridization patterns confirmed the similarity between the mating-type (-) strains and showed some heterogeneity among the mating-type (+) strains. Linkage was found between genes pyrF and chs3 in all the strains, between the gene leuA and the rDNA in all mating-type (-) strains and ATCC 1216b (+), and between chs2 and the rDNA in CBS 969.68 (+). This is the first time that gene linkage in M. circinelloides has been reported, but in some cases the linkage relationships obtained are strain-dependent.
Asunto(s)
Electroforesis en Gel de Campo Pulsado/métodos , Mucor/química , Mucor/genética , Southern Blotting , Cromosomas Fúngicos/genética , ADN Ribosómico/genética , Prueba de Complementación Genética , Heterogeneidad Genética , Ligamiento Genético , Polimorfismo Genético , ReproducciónRESUMEN
Fusarium oxysporum Schechtend.:Fr. f. sp. phaseoli J. B. Kendrick & W. C. Snyder (FOP) is the causal agent of the common bean (Phaseolus vulgaris L.) disease known as Fusarium wilt or Fusarium yellows. FOP has been reported from the Castilla y Leon region in Spain, where it is a serious problem on most commercial bean cultivars (1). Five FOP isolates from Spain (AB-6, AB-111, AB-112, AS-1, and AS-4) obtained from J. M. Díaz-Mínguez and the isolate FOP-CO1 (ATCC 90245) from Colorado were tested for pathogenicity on two American lines: Pinto U.I. 114, considered as a universal susceptible check, and Flor de Mayo, a Mexican landrace. Seedlings were root-clip inoculated and evaluated according to the CIAT 1 to 9 severity scale, in which 1 to 3 = resistant, 3.1 to 6 = intermediate, and 6.1 to 9 = susceptible. Three inoculum concentrations were tested: 104, 105, and 106 conidia/ml. AS-1 and AS-4 produced resistant reactions in both U.I. 114 (1.5 to 2.1 and 1.2 to 1.5) and Flor de Mayo (1.9 to 2.3 and 1.4), respectively, at all inoculum concentrations. Susceptible reactions to AB-6 (7.3 to 8.9), AB-111 (7.6 to 8.9), AB-112 (7.5 to 7.9), and FOP-CO1 (8.1 to 8.6) were observed in Pinto U.I. 114, regardless of inoculum concentration, although severity ratings increased as the inoculum concentration was increased. Flor de Mayo exhibited a susceptible reaction (6.7 to 7.8) to FOP-CO1 at all inoculum concentrations tested, and intermediate reactions to AB-6 (4.9), AB-111 (5.4), and AB-112 (5.0) at the lowest inoculum concentration. Susceptible reactions (7 to 8.2 for AB-6; 6.8 to 7.0 for AB-111, and 7.3 to 7.7 for AB-112) occurred with higher inoculum concentrations, and severity ratings increased as the inoculum concentration increased. Recently, FOP isolates from Greece and Italy were recognized as each belonging to different pathogenic races (2); consequently, more research on the Spanish isolates (AB-6, AB-111, and AB-112) is needed to determine if they are similar to the races reported from the Mediterranean Basin or should be classified as a new FOP race (s). References: (1) J. M. Díaz-Mínguez et al. Plant Dis. 80:600, 1996. (2) S. L. Woo et al. Phytopathology 86:966, 1996.
RESUMEN
A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene of Mucor circinelloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe. The complete nucleotide sequence of the M. circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis. The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P. blakesleeanus. Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart. The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes. Northern-blot hybridization revealed the pyrG transcript to be approx. 1.0 kb. The M. circinelloides pyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coli when inserted between bacterial expression signals. Additionally, the genomic clone complemented the M. circinelloides pyrG4 mutation. When an M. circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per micrograms DNA and per 10(6) viable protoplasts.
Asunto(s)
Mucor/enzimología , Orotidina-5'-Fosfato Descarboxilasa/genética , Transformación Genética/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Prueba de Complementación Genética , Intrones/genética , Datos de Secuencia Molecular , Mucor/genética , Mutación/genética , Orotidina-5'-Fosfato Descarboxilasa/química , Plásmidos/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
The leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus alpha-IPM (isopropylmalate) isomerase.
Asunto(s)
Genes Fúngicos , Técnicas Genéticas , Hidroliasas/genética , Mucor/genética , Phycomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Regulación Fúngica de la Expresión Génica , Biblioteca Genómica , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Transformación GenéticaRESUMEN
The pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5'-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal "AATAAA". The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.
Asunto(s)
Genes Fúngicos , Orotidina-5'-Fosfato Descarboxilasa/genética , Phycomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón/genética , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Hongos/genética , Humanos , Intrones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Phycomyces/enzimología , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaAsunto(s)
2-Isopropilmalato Sintasa/genética , Leucina/genética , Mucorales/genética , Oxo-Ácido-Liasas/genética , Phycomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , Genes Fúngicos , Datos de Secuencia Molecular , Phycomyces/enzimología , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.