RESUMEN
Grapevine trunk diseases cause serious economic losses to grape growers worldwide. The identification of the causal fungi is critical to implementing appropriate management strategies. Through a culture-based approach, we identified the fungal species composition associated with symptomatic grapevines from wine grapes in southeastern Washington and table grapes in the southern San Joaquin Valley of California, two regions with contrasting winter climates. Species were confirmed through molecular identification, sequencing two to six gene regions per isolate. Multilocus phylogenetic analyses were used to identify novel species. We identified 36 species from 112 isolates, with a combination of species that are new to science, are known causal fungi of grapevine trunk diseases, or are known causal fungi of diseases of other woody plants. The novel species Cadophora columbiana, Cytospora macropycnidia, Cytospora yakimana, and Sporocadus incarnatus are formally described and introduced, six species are newly reported from North America, and grape is reported as a new host for three species. Six species were shared between the two regions: Cytospora viticola, Diatrype stigma, Diplodia seriata, Kalmusia variispora, Phaeoacremonium minimum, and Phaeomoniella chlamydospora. Dominating the fungal community in Washington wine grape vineyards were species in the fungal families Diatrypaceae, Cytosporaceae and Sporocadaceae, whereas in California table grape vineyards, the dominant species were in the families Diatrypaceae, Togniniaceae, Phaeomoniellaceae and Hymenochaetaceae. Pathogenicity tests demonstrated that 10 isolates caused wood discoloration similar to symptomatic wood from which they were originally isolated. Growth rates at temperatures from 5 to 35°C of 10 isolates per region, suggest that adaptation to local climate might explain their distribution.
RESUMEN
In California vineyards, spore dispersal of fungi that cause grapevine trunk diseases Botryosphaeria dieback and Eutypa dieback occurs with winter rains. Spores infect through pruning wounds made to the woody structure of the vine in winter. Better timing of preventative practices that minimize infection may benefit from routine spore-trapping, which could pinpoint site-specific time frames of spore dispersal. To speed pathogen detection from environmental spore samples, we identified species-specific PCR primers and protocols. Then we compared the traditional culture-based method versus our new DNA-based method.â¢PCR primers for Botryosphaeria-dieback pathogen Neofusicoccum parvum and Eutypa-dieback pathogen Eutypa lata were confirmed species-specific, through extensive testing of related species (in families Botryosphaeriaceae and Diatrypaceae, respectively), other trunk-disease pathogens, and saprophytic fungi that sporulate in vineyards.â¢Consistent detection of N. parvum was achieved from spore suspensions used fresh or stored at -20°C, whereas consistent detection of E. lata was achieved only with a new spore-lysis method, using zirconia/silica beads in a FastPrep homogenizer (MP Biomedicals; Solon, Ohio, USA), and only from spore suspensions used fresh. Freezing E. lata spores at -20°C made detection inconsistent.â¢From environmental samples, spores of E. lata were detected only via PCR, whereas spores of N. parvum were detected both via PCR and in culture.
RESUMEN
Eutypa dieback of grapevine is caused by Eutypa lata in production areas with Mediterranean climates in California, Australasia, Europe, and South Africa. Eutypa dieback has also been described in the colder, eastern North American vineyards where cultivars adapted from native Vitis spp. (e.g., Vitis × labruscana 'Concord') are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vineyards showing symptoms of dieback in the northeastern United States (Connecticut, Massachusetts, Michigan, New York, Ohio, and Rhode Island) and Ontario, Canada revealed that vineyards were mainly infected by Eutypa spp. other than E. lata. Multigene phylogenies (internal transcribed spacer ribosomal DNA, ß-tubulin, and RNA polymerase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. laevata. Eutypa sp. was recovered from 56% of the vineyards examined, whereas E. laevata and E. lata were less far common (17 and 6%, respectively). Fruiting body morphology and spore dimensions supported phylogenetic separation of the three taxa. Pathogenicity tests conducted on Vitis vinifera 'Chardonnay' in the greenhouse and in the field verified that all three species were able to cause wood canker and to infect pruning wounds, respectively.
RESUMEN
In eastern North America, Phomopsis cane and leaf spot, caused by Phomopsis viticola, is a foliar disease of grape but, in the Mediterranean climate of western North America, P. viticola is primarily associated with wood cankers, along with other Diaporthe spp. To determine the identity of wood-infecting Diaporthe spp. in eastern North America, 65 isolates were cultured from 190 wood-canker samples from 23 vineyards with a history of Phomopsis cane and leaf spot. Identification of 29 representative isolates was based initially on morphology, followed by phylogenetic analyses of DNA sequences of the ribosomal DNA internal transcribed spacer region, elongation factor subunit 1-α, and actin in comparison with those of type specimens. Three species were identified: P. viticola, P. fukushii, and Diaporthe eres. Inoculations onto woody stems of potted Vitis labruscana 'Concord' and V. vinifera 'Chardonnay' showed that D. eres and P. fukushii were pathogenic (mean lesion lengths of 7.4 and 7.1 mm, respectively, compared with 3.5 mm for noninoculated controls) but significantly less so than wood-canker and leaf-spot isolates of P. viticola (13.5 mm). All three species infected pruning wounds of Concord and Chardonnay in the field. Our finding of pathogenic, wood-infecting Diaporthe spp. in all 23 vineyards suggests a frequent co-occurrence of the foliar symptoms of Phomopsis cane and leaf spot and wood cankers, although the latter are not always due to P. viticola.
RESUMEN
Investigating the dispersal of the root-pathogenic fungus Armillaria mellea is necessary to understand its population biology. Such an investigation is complicated by both its subterranean habit and the persistence of genotypes over successive host generations. As such, host colonization by resident mycelia is thought to outcompete spore infections. We evaluated the contributions of mycelium and spores to host colonization by examining a site in which hosts pre-date A. mellea. Golden Gate Park (San Francisco, CA, USA) was established in 1872 primarily on sand dunes that supported no resident mycelia. Genotypes were identified by microsatellite markers and somatic incompatibility pairings. Spatial autocorrelation analyses of kinship coefficients were used to infer spore dispersal distance. The largest genotypes measured 322 and 343 m in length, and 61 of the 90 total genotypes were recovered from only one tree. The absence of multilocus linkage disequilibrium and the high proportion of unique genotypes suggest that spore dispersal is an important part of the ecology and establishment of A. mellea in this ornamental landscape. Spatial autocorrelations indicated a significant spatial population structure consistent with limited spore dispersal. This isolation-by-distance pattern suggests that most spores disperse over a few meters, which is consistent with recent, direct estimates based on spore trapping data.
Asunto(s)
Armillaria/genética , Armillaria/patogenicidad , Enfermedades de las Plantas/microbiología , Demografía , Frecuencia de los Genes , Genética de Población , Desequilibrio de Ligamiento , Repeticiones de Microsatélite , San FranciscoRESUMEN
Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.
Asunto(s)
Agrobacterium tumefaciens/genética , Armillaria/genética , Genética Microbiana/métodos , Recombinación Genética , Cruzamientos Genéticos , ADN de Hongos/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Selección Genética , Análisis de Secuencia de ADNRESUMEN
Slow and unreliable infection in the greenhouse has been a barrier to research on Armillaria root disease. The existing infection assay takes 7-18 months for detectable infection, during which time the inoculum often dies, resulting in unequal challenge among plants. Because symptom expression and mortality are rare, presence or absence of infection, determined by culturing, is the only datum derived from the existing infection assay. This limits both routine comparisons of strain virulence and complex investigations of pathogenesis, neither of which have been done for Armillaria mellea. We tested a new infection assay, in which grape rootstocks growing in tissue culture medium are inoculated, and compared to rootstocks previously characterized from the existing infection assay as tolerant (Freedom) or susceptible (3309C). Culture media of 25 plants per rootstock was inoculated and five plants per rootstock were harvested 0, 2, 4, 6, and 8 weeks postinoculation; the experiment was completed twice. Confocal microscopy and quantitative PCR (Q-PCR) were used to quantify infection. Roots were treated with WGA-AlexaFluor488, hyphae and roots were scanned on green and red channels on a confocal microscope, and percent root colonization was quantified. A fungal gene (EF1?) was determined to have a single copy in A. mellea, and both EF1? and a single-copy grape gene (UFGT) were amplified by Q-PCR; fungal DNA: plant DNA served as a measure of fungal biomass. Armillaria was detected by culture, microscopy, and Q-PCR starting 2 weeks postinoculation from all inoculated plants, demonstrating that the new infection assay is rapid and plants do not escape infection. Our findings of higher percent root colonization (as measured by microscopy) of 3309C than Freedom at all harvests (P<0.0001), consistently higher fungal biomass (as measured by Q-PCR) of 3309 than Freedom, and a significant positive correlation between percent root colonization and fungal biomass (P=0.01) suggests that the quantitative methods of our new assay give similar results to the qualitative method of the existing infection assay.
Asunto(s)
Armillaria/patogenicidad , Biomasa , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Reacción en Cadena de la Polimerasa/métodos , Vitis/microbiología , Armillaria/genética , Armillaria/metabolismo , ADN de Hongos/análisis , ADN de Hongos/genética , ADN de Plantas/análisis , ADN de Plantas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Microscopía Confocal , Factor 1 de Elongación Peptídica/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Tiempo , Vitis/genética , Vitis/metabolismoRESUMEN
Abstract Efforts were made to develop a human adipocyte model that is useful for toxicological studies in vitro. For this purpose, a stem cell line derived from human bone marrow cells, originally from an adult, was induced to differentiate towards adipocytes by treating them with insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine for 3 d, followed by additional incubation for 3 d in Dulbecco's modified Eagle's medium supplemented with insulin only. In most cases, thus differentiated cells through such one cycle of differentiation treatment were further subjected to the second cycle of differentiation. The resulting 2-cycle differentiated cells were found to exhibit many characteristics of typical adipocytes. Dioxin (TCDD), when added at the beginning of their treatment with differentiation-inducing hormone cocktail, clearly prevented them from becoming adipocytes, as in the case of TCDD-treated 3T3-L1 cells. Furthermore, TCDD, even when administered to previously differentiated human mesenchymal stem cells (hMSC) adipocytes, consistently induced the sign of inflammatory responses during the early period of TCDD action (24 h), which was followed by gradual loss of adipocyte-specific markers during the 5-d incubation period. In conclusion, hMSC-derived adipocytes appear to offer a promising human cell model suited for future toxicological studies.
Asunto(s)
Adipocitos/citología , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Dibenzodioxinas Policloradas/toxicidad , Células de la Médula Ósea , Técnicas de Cultivo de Célula , Ecotoxicología , Contaminantes Ambientales/toxicidad , Humanos , Teratógenos/toxicidad , Pruebas de ToxicidadRESUMEN
BACKGROUND: One of the outcomes positively associated with dioxin exposure in humans is type 2 diabetes. OBJECTIVES: This study was conducted in order to find the molecular biological evidence for the diabetogenic action of dioxin in adipose samples from Vietnam veterans. METHODS: We obtained 313 adipose tissue samples both from Vietnam veterans who were exposed to dioxin (Operation Ranch Hand) and from comparison veterans who served in Southeast Asia with no record of dioxin exposure. We conducted quantitative reverse-transcribed polymerase chain reaction studies on selected marker mRNAs from these samples. RESULTS: We found the most sensitive and reliable molecular indicator of dioxin-induced diabetes to be the ratio of mRNA of glucose transporter 4 (GLUT4) and nuclear transcription factor kappa B (NFkappaB), a marker of inflammation. This ratio showed significant correlations to serum dioxin residues and to fasting glucose among those in the Ranch Hand group and, surprisingly, even in the comparison group, who have low levels of dioxin comparable to the general public. Such a correlation in the comparison group was particularly significant among those with known risk factors such as obesity and family history of diabetes. CONCLUSIONS: These results show that the GLUT4:NFkappaB ratio is a reliable marker for the diabetogenic action of dioxin, particularly at very low exposure levels that are not much higher than those found in the general public, implying a need to address current exposure levels.
Asunto(s)
Diabetes Mellitus Tipo 2/etiología , Dioxinas/toxicidad , Contaminantes Ambientales/toxicidad , Transportador de Glucosa de Tipo 4/metabolismo , FN-kappa B/metabolismo , Ácido 2,4,5-Triclorofenoxiacético/toxicidad , Ácido 2,4-Diclorofenoxiacético/toxicidad , Tejido Adiposo/metabolismo , Agente Naranja , Biomarcadores/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Defoliantes Químicos/toxicidad , Diabetes Mellitus Tipo 2/metabolismo , Dioxinas/sangre , Monitoreo del Ambiente , Contaminantes Ambientales/sangre , Genes src/genética , Transportador de Glucosa de Tipo 4/genética , Humanos , Masculino , Epidemiología Molecular , FN-kappa B/genética , Dibenzodioxinas Policloradas/toxicidad , ARN Mensajero/metabolismo , Estados Unidos , Veteranos , Guerra de VietnamRESUMEN
Diflubenzuron (DIMILIN) is a powerful insecticidal chemical which has been known for many years to inhibit chitin synthesis in vivo in insects and related arthropod species. However, its action mechanism has remained unresolved partly because of its inaction on any of the enzymes involved in chitin synthesis in vitro. Based on our previous work (Diflubenzuron affects gamma-thioGTP stimulated Ca2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L. Insect Biochem. Mol. Biol. 24 (1994) 1009) showing that diflubenzuron inhibits Ca2+ uptake by vesicles obtained from the integument of American cockroach, Periplaneta americana (L.), in vitro, we tested the hypothesis that the action site of diflubenzuron is an ABC (ATP binding cassette) transporter, probably a sulfonylurea-sensitive transporter. Glibenclamide, one of the most commonly used sulfonylureas for type II diabetes treatment, was the positive control. When given to immature insects, glibenclamide clearly caused toxicity, with symptoms indicating molting abnormality comparable to diflubenzuron. Its LD50 (0.472 microg/nymph) was approximately 2.8 times the value obtained for diflubenzuron (0.17 microg/nymph, topical) in German cockroach, Blattella germanica (L.). However, in terms of the inhibitory activities on chitin synthesis, in isolated integuments glibenclamide showed an identical potency to diflubenzuron in B. germanica nymphs. A competitive binding assay with [3H]-glibenclamide and unlabeled diflubenzuron clearly established that the latter is capable of competitively displacing the former radioligand. The KD values observed for vesicles prepared from fruit fly larvae, Drosophila melanogaster M., were 44.9 nM for glibenclamide and 65.0 nM for diflubenzuron, respectively. Furthermore, glibenclamide was found to affect Ca2+ uptake by isolated cuticular vesicles from B. germanica in a manner very similar to diflubenzuron. These results support our conclusion that the sulfonylurea receptor (SUR) is the target of diflubenzuron in inhibition of chitin synthesis in these two insect species.