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ABSTRACT Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the beta-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.
RESUMEN
Potato early dying (PED), caused by Verticillium dahliae, is a chronic yield-limiting disease of potato (Solanum tuberosum). In this study, we describe the colonization dynamics of V. dahliae in two potato cultivars with varying responses to PED. We utilized a quantitative real-time polymerase chain reaction (Q-PCR) assay to assess the colonization and spatial progression of V. dahliae in cvs. Ranger Russet (moderately resistant) and Russet Norkotah (highly susceptible). Ninety plants per cultivar were inoculated with a conidial suspension in the greenhouse. Every 2 weeks until week 10, we collected basal samples from 15 plants, and repeatedly sampled the growing apices of another 15 plants. The mean infection coefficient (IC) values in the basal and apical samples were significantly lower in cv. Ranger Russet at all five sampling dates. The pathogen was detected in basal samples of both cultivars by week 2, and in apical samples of cv. Russet Norkotah at week 4 and of cv. Ranger Russet at week 6. Colonization of cv. Russet Norkotah consistently increased in apical and basal samples during the 10 weeks, while it plateaued after week 6 in cv. Ranger Russet. Differences in response to PED appear associated with the speed of colonization and the establishment of a higher population density by V. dahliae in the plant.
RESUMEN
Verticillium wilt (Vw), caused by the soilborne fungi Verticillium dahliae and V. albo-atrum, is an important disease of potato (Solanum tuberosum). Host plant resistance is a promising method of Vw control. Culture-based methods that quantify the pathogen in host tissue often are used for Vw resistance screening. To evaluate the processing time, accuracy, and precision of these methods, 46 clones were planted in a field naturally infested with V. dahliae to collect data on visual disease symptoms, pathogen colonization, and yield. In 2002, disease severity explained 4.34% of the variability of yield loss, but the linear relationship between stem colonization and yield loss was not significant. In 2003, stem colonization explained 57.5% of the variability of yield loss, whereas disease severity explained 1.7% of the variability of yield loss. Correlations comparing clone ranks from repeated pathogen measurements indicated that culturing sap from individual stems or bulked stems generated more repeatable clone rankings than culturing dried stems. Clone rankings were more repeatable between years if pathogen measurements were made earlier in the growing season. The results indicate a need to characterize the effect of the environment on the relationship among pathogen population sizes in planta, disease symptoms, and yield loss.
RESUMEN
Verticillium wilt is a serious disease in potato and is caused primarily by the soilborne fungi Verticillium dahliae and V. albo-atrum. Host plant resistance to the disease offers an option for long-term, inexpensive, and environmentally sound control. High levels of resistance to stem colonization have been identified in two diploid hybrids between the cultivated potato and wild Solanum spp. An intercross between the two clones produced a 3:1 ratio of resistant to susceptible clones. A cross between a susceptible clone and one of the resistant clones also produced a 3:1 resistant:susceptible ratio. These data can be explained by a two-gene model, in which dominant alleles of both genes must be present to confer resistance. The two-gene model also explains data from previous research with wild Solanum spp. A simple mode of inheritance should improve the probability of producing resistant offspring when resistant hybrids are used as parents in a breeding program.
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Wild species of Solanum are excellent sources of disease resistance genes that may be incorporated into S. tuberosum through breeding. This study was initiated to determine whether multiple forms of disease resistance could be identified in interspecific Solanum hybrids. Thirty-two clones were evaluated for resistance to soft rot, common scab, black scurf, Verticillium wilt, and early blight. Most of the clones originated from populations that were not initially selected for disease resistance traits. Comparisons with the cultivars Atlantic, Russet Norkotah, and Russet Burbank indicated that all clones were more resistant than at least one cultivar for at least one disease resistance trait. Clone C545, which exhibited improved resistance to soft rot, scab, pitted scab, early dying disease, and early blight, appears to be an especially valuable source of disease resistance. The use of interspecific hybridization at the diploid level, combined with sexual polyploidization to return to the tetraploid level, provides a method to introduce multiple forms of disease resistance into advanced clones.
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Four similar growth chamber experiments were conducted to test the hypothesis that the initial population density (Pi) of Pratylenchus penetrans influences the severity of interactive effects of P. penetrans and Verticillium dahliae on shoot growth, photosynthesis, and tuber yield of Russet Burbank potato. In each experiment, three population densities of P. penetrans with and without concomitant inoculation with V. dahliae were compared with nematode-free controls. The three specific Pi of JR penetrans tested varied from experiment to experiment but fell in the ranges 0.8-2.5, 1.8-3.9, 2.1-8.8, and 7.5-32.4 nematodes/cm(3) soil. Inoculum of V. dahliaewas mixed into soil, and the assayed density was 5.4 propagules/gram dry soil. Plants were grown 60 to 80 days in a controlled environment. Plant growth parameters in two experiments indicated significant interactions between P. penetrans and V. dahliae. In the absence of V. dahliae, P. penetrans did not reduce plant growth and tuber yield below that of the nematode-free control or did so only at the highest one or two population densities tested. In the presence of K dahliae, the lowest population density significantly reduced shoot weight and photosynthesis in three and four experiments, respectively. Higher densities had no additional effect on shoot weight and caused additional reductions in photosynthesis in only one experiment. Population densities of 0.8 and 7.5 nematodes/cm(3) soil reduced tuber yield by 51% and 45%, whereas higher densities had no effect or a 15% additional effect, respectively. These data indicate that interactive effects between P. penetrans and V. dahliae on Russet Burbank potato are manifested at P. penetrans population densities less than 1 nematode/cm(3) soil and that the nematode population density must be substantially higher before additional effects are apparent.
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ABSTRACT The effects of solitary and concurrent infection by Pratylenchus pene-trans and Verticillium dahliae on gas exchange of Russet Burbank potato (Solanum tuberosum) were studied in growth chamber experiments. Treatments were P. penetrans at low, medium, and high density; V. dahliae alone at one initial density; the combination of the nematode at these three densities and V. dahliae; and a noninfested control. Gas exchange parameters of leaf cohorts of different ages in the different treatments were repeatedly measured with a Li-Cor LI-6200 portable photosynthesis system. At 45 days after planting, joint infection significantly reduced net photosynthesis, stomatal conductance, and transpiration of 1- to 25-day-old leaf cohorts. Intercellular CO(2) levels were significantly increased by co-infection, especially in older leaves. The synergistic effect of co-infection on gas exchange parameters was greater in the oldest cohort than in the youngest cohort. No consistent effects on leaf gas exchange parameters were observed in plants infected by the nematode or the fungus alone. The relationship between the assimilation rate and stomatal conductance remained linear regardless of solitary or concomitant infection, indicating that stomatal factors are primarily responsible for regulating photosynthesis. The significant reduction of gas exchange in leaves of co-infected plants without reduction in intercellular CO(2) concentrations suggests that nonstomatal factors also play a role when both organisms are present.
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ABSTRACT The interactive effects of concomitant infection by the nematode Pratylenchus penetrans and the fungus Verticillium dahliae on symptom expression in Russet Burbank potato was studied in growth chamber experiments. Treatments were P. penetrans at three initial densities, V. dahliae at one inoculum density, the combination of the nematode at these three densities and the fungus, and a noninfested control. Gas exchange was measured nondestructively in leaf cohorts of different ages, one to three times weekly, with a LI-COR portable photosynthesis system. The single-pathogen treatments had no effect on assimilation or transpiration rates, but joint infection had a significant impact. In concomitant infection, photosynthesis was impaired more than transpiration, so estimates of leaf health were based on carbon assimilation rates only. Reductions in assimilation rate were apparent before the onset of visual symptoms. Assimilation rates decreased as much as 44% in the top, and newest, leaves of concomitantly infected plants, compared to rates in control plants. Even so, the health of newly produced leaves did not become progressively worse through time. With light use efficiency less than 0.20 mol of CO(2) fixed per mol of photosynthetically active radiation used as the criterion for disease incidence, disease progressed acropetally from the oldest to the youngest leaves. In plants infected with P. penetrans (0.8 nematodes per cm(3) of soil) in combination with V. dahliae, all leaves in cohorts 1 and 2 were symptomatic by 45 days after planting, and leaves in cohorts 3 to 6 became symptomatic at weekly intervals thereafter. For the control and single-pathogen treatments, the first time that light use efficiency fell below 0.20 in all leaves in cohort 1 was 71 days after planting. Concomitant infection reduced leaf life span by about 3 weeks. Both visual and physiological symptom expression were invariant to differences in initial nematode inoculum densities ranging from 0.8 to 2.5 nematodes per cm(3) of soil in one experiment and from 1.3 to 4.1 nematodes per cm(3) of soil in a second experiment.
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Soil column studies were conducted to investigate the influence of soil water content and temperature on the efficacy of metham-sodium and its degradation product methyl isothiocyanate against Verticillium dahliae. The viability of the microsclerotia (MS) of the fungus in the top 30 cm of fumigated and control columns was measured. Temperatures for studies were 2 or 22°C, and the soil water content, expressed as soil matric potential, varied from -23 (wet), -113 (moist), to -2485 J/kg (dry). There was a significant interaction of soil water content and temperature on the efficacy of metham-sodium against V. dahliae MS. For the low soil temperature (2°C) the fumigant was more effective against MS of the fungus in wet than in moist or dry soil Soil water content did not affect fungicidal activity of metham-sodium when the soil columns were maintained at 22°C. These results suggest that the fumigant has a greater efficacy against V. dahliae in wet/cold soil conditions compared to the other conditions tested. Consequently, it was recommended that metham-sodium be applied to fields by chemigation in late fall or early spring to obtain efficacious results.
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A pair of conserved PCR primers, designated NMS1 and NMS2, that amplify a region in the mitochondrial small rRNA gene region were designed for fungi belonging to the class Ascomycetes. These primers were tested with members of eight fungal genera (Aspergillus, Fusarium, Magnaporthe, Mycospharella, Neurospora, Saccharomyces, Sclerotinia, Verticillium) and 10 Verticillium species (Verticillium albo-atrum, Verticillium chlamydosporium, Verticillium cinnebarium, Verticillium dahliae, Verticillium fungicola, Verticillium lecanii, Verticillium lateritium, Verticillium nigrescens, Verticillium psaliotae, and Verticillium tricorpus). The primers were also tested with 35 isolates of V. dahliae obtained from diverse geographic areas and diverse hosts. The results of a restriction fragment length polymorphism analysis of the region amplified by the primers differentiated the genera examined and the results of a DNA sequence analysis of the amplified region differentiated the Verticillium species. Two Fusarium species were also differentiated by the results of the restriction fragment length polymorphism analysis. On the basis of the nucleotide sequences of the amplified regions, we obtained a pair of PCR primers that could be used to differentiate V. dahliae from the other fungal isolates tested, including V. albo-atrum, a closely related plant-pathogenic species. The V. dahliae-specific PCR primer may aid in more rapid and specific detection of the pathogen directly in plant and/or soil samples. PCR primers NMS1 and NMS2 may be used as potential mitochondrial markers for studying fungal cytoplasmic inheritance of ascomycetes and for identifying DNA probes that are informative at or below the genus level.
Asunto(s)
Cartilla de ADN , ADN de Hongos/análisis , Fusarium/genética , Hongos Mitospóricos/genética , Polimorfismo de Longitud del Fragmento de Restricción , Aspergillus/clasificación , Aspergillus/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Mitocondrial/análisis , Fusarium/clasificación , Genes Fúngicos/genética , Hongos Mitospóricos/clasificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN de Hongos/genética , ARN Ribosómico/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Young, visually symptomless leaves from potato (Solanum tuberosum) plants infected with Verticillium dahliae exhibited reduced carbon assimilation rate, stomatal conductance, and intercellular CO(2), but no increase in dark respiration, no change in the relationship between carbon assimilation rate versus intercellular CO(2), and no change in light use efficiency when intercellular CO(2) was held constant. Therefore, the initial decrease in photosynthesis caused by V. dahliae was caused by stomatal closure. Errors in the intercellular CO(2) calculation caused by uneven distribution of carbon assimilation rate across the leaf were tested by (14)CO(2) autoradiography. Patchiness was found at a low frequency. Low stomatal conductance was correlated with low leaf water potentials. Infection did not affect leaf osmotic potentials.